Treatment of intervertebral disc degeneration using human umbilical cord tissue-derived cells and hydrogels
阅读说明：本技术 使用人脐带组织来源细胞和水凝胶治疗椎间盘退变 (Treatment of intervertebral disc degeneration using human umbilical cord tissue-derived cells and hydrogels ) 是由 B.C.克拉梅 L.J.布朗 A.J.基姆 于 2012-05-17 设计创作，主要内容包括：本发明提供了治疗患有与IVD退变相关的疾病或病症的患者的方法。所述方法包括施用从人脐带组织获得的细胞或施用包含此类细胞或由此类细胞和水凝胶制备的药物组合物。在一些实施例中,施用所述细胞促进所述患者中的退变IVD组织的修复和再生。本发明还提供了用于本发明方法的药物组合物以及用于实践所述方法的试剂盒。(The present invention provides methods of treating a patient having a disease or disorder associated with IVD degeneration. The methods comprise administering cells obtained from human umbilical cord tissue or administering a pharmaceutical composition comprising such cells or prepared from such cells and a hydrogel. In some embodiments, administering the cells promotes repair and regeneration of degenerated IVD tissue in the patient. The invention also provides pharmaceutical compositions for use in the methods of the invention and kits for practicing the methods.)
1. Use of a pharmaceutical composition comprising fibrinogen, thrombin and an isolated homogenous population of cells obtained from human umbilical cord tissue in the manufacture of a medicament for improving the cellular structure and architecture of an intervertebral disc in a patient suffering from degeneration of the intervertebral disc, wherein the umbilical cord tissue is substantially free of blood, and wherein the isolated homogenous population of cells is capable of self-renewal and expansion in culture and has the potential to differentiate without expressing CD117 or telomerase.
2. The use of claim 1, wherein the isolated population of cells has one or more of the following properties:
(a) expressing plasma membrane protein, chemokine receptor ligand 3, and/or granulocyte chemotactic protein;
(b) does not produce CD31, CD34 and HLA-DR;
(c) expresses increased levels of oxidized interleukin 8 and reticulon 1 relative to human fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells; and
(d) express CD10, CD13, CD44, CD73 and CD 90.
3. The use of claim 1 or 2, wherein the pharmaceutical composition is formulated for administration by injection.
4. The use according to claim 1 or 2, wherein the pharmaceutical composition further comprises at least one other cell type and/or at least one agent.
5. The use of claim 4, wherein the at least one agent is a trophic factor.
6. The use of claim 5, wherein the trophic factor is selected from the group consisting of TGF- β, GDF-5, PDGF-BB, and TIMP 1.
7. The use of claim 4, wherein the at least one other cell type is engineered to express at least one exogenous gene product.
8. The use of claim 4, wherein the exogenous gene product is a trophic factor.
9. The use of claim 1 or 2, wherein the pharmaceutical composition is a formulation for administration into a degenerated intervertebral disc.
10. The use of claim 9, wherein the pharmaceutical composition is a formulation for administration into the nucleus pulposus or into the annulus fibrosus of the intervertebral disc.
11. The use of claim 1 or 2, further comprising inducing the isolated homogenous population of cells obtained from human umbilical cord tissue to at least partially differentiate in vitro.
12. The use of claim 11, wherein the isolated homogenous population of cells is induced to differentiate into cells exhibiting an annulus fibrosus cell phenotype or into cells exhibiting a nucleus pulposus cell phenotype.
13. The use of claim 1, wherein the composition comprises 6.8-10.6mg/ml fibrinogen and 0.4-0.6U/ml thrombin.
14. Use of fibrinogen, thrombin and an isolated homogenous population of cells obtained from human umbilical cord tissue in the preparation of a medicament for improving the cellular structure and architecture of an intervertebral disc in a patient suffering from degeneration of the intervertebral disc, wherein the umbilical cord tissue is substantially free of blood, and wherein the isolated homogenous population of cells is capable of self-renewal and expansion in culture and has the potential to differentiate, but does not express CD117 or telomerase.
15. The use of claim 14, wherein the isolated population of cells has one or more of the following properties:
(a) express plasma membrane protein, chemokine receptor ligand 3, and granulocyte chemotactic protein;
(b) does not produce CD31, CD34 and HLA-DR;
(c) expresses increased levels of interleukin 8 and reticulon 1 relative to human fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells; and
(d) express CD10, CD13, CD44, CD73 and CD 90.
16. The use of claim 14 or 15, wherein the isolated homogenous population of cells and hydrogel are formulated for administration by injection.
17. The use of claim 14 or 15, wherein hydrogel and the isolated homogenous population of cells obtained from human umbilical cord tissue are provided as a kit.
18. The use of claim 14 or 15, wherein the isolated homogenous population of cells is within an implantable device.
19. The use of claim 14 or 15, further comprising at least one other cell type.
20. The use of claim 19, wherein the at least one other cell type is engineered to express at least one exogenous gene product.
21. The use of claim 20, wherein the exogenous gene product is a trophic factor.
22. The use of claim 21, wherein the exogenous gene product modulates the expression of one or more extracellular matrix proteins.
23. The use of claim 14 or 15, further comprising at least one agent.
24. The use of claim 23, wherein the at least one agent is a trophic factor.
25. The use of claim 24, wherein the trophic factor is selected from TGF- β, GDF-5, PDGF-BB, and TIMP 1.
26. The use of claim 25, wherein the trophic factor has a trophic effect on the isolated homogenous population of cells obtained from human umbilical cord tissue.
27. The use of claim 14 or 15, wherein the isolated homogenous population of cells and hydrogel are administered into a degenerated intervertebral disc.
28. The use of claim 27, wherein the isolated homogenous population of cells and hydrogel are formulated for administration into a nucleus pulposus of the intervertebral disc.
29. The use of claim 27, wherein the isolated homogenous population of cells and hydrogel are formulated for administration into the annulus fibrosus of the intervertebral disc.
30. The use of claim 14 or 15, wherein the isolated homogenous population of cells obtained from human umbilical cord tissue is induced to at least partially differentiate in vitro.
31. The use of claim 30, wherein the isolated homogenous population of cells is induced to differentiate into cells exhibiting an annulus fibrosus cell phenotype or into cells exhibiting a nucleus pulposus cell phenotype.
32. The use according to claim 14, wherein the use comprises 6.8-10.6mg/ml fibrinogen and 0.4-0.6U/ml thrombin.
Technical Field
The present invention relates generally to the field of cell-based therapies. In some aspects, the invention relates to the use of umbilical cord tissue-derived cells to treat a disease or disorder associated with degeneration of an intervertebral disc.
Background
Throughout this specification, various publications are referenced, including patents, published patent applications, technical literature, and academic literature. Each of these cited publications is herein incorporated by reference in its entirety for all purposes.
Lower back pain is one of the most common disabilities, causing significant physical and emotional discomfort to the patient. Degeneration of the intervertebral disc (IVD) structure is one of the major causes of lower back pain. The IVD is formed by a fibrous outer annulus fibrosis surrounding a softer gelatinous nucleus pulposus. The fibers of the annulus fibrosus attach to the endplates of the vertebral bodies of the spinal cord and surround the nucleus pulposus, forming an isotonic environment. Under axial load, the nucleus compresses and transfers this load radially to the annulus. The laminar nature of the annulus fibrosus provides it with high tensile strength, thus allowing it to expand radially in response to this transmitted load.
In healthy IVD, the cells within the nucleus pulposus form only about one percent of the disc tissue by volume. These cells produce an extracellular matrix (ECM) containing a high percentage of proteoglycans. Proteoglycans contain water-retaining sulfated functional groups, thereby providing the nucleus pulposus with its cushioning properties. The nucleus pulposus cells also secrete small amounts of cytokines and Matrix Metalloproteinases (MMPs), which help regulate the metabolism of the nucleus pulposus cells.
In some cases of IVD disease, gradual degeneration of IVD is caused by mechanical instability of other parts of the spine. Under these circumstances, increased load and pressure on the nucleus pulposus can cause cells (or invading macrophages) within the intervertebral disc to release higher than normal amounts of the above cytokines. In other cases of IVD disease, genetic factors or apoptosis can cause a decrease in the number of intervertebral disc cells and/or release toxic amounts of cytokines and MMPs. In some cases, the pumping action of the disc may fail (due to, for example, a decrease in the concentration of proteoglycans within the nucleus pulposus), thereby slowing the rate of nutrient flow into the disc and the rate of waste products flow out of the disc. This reduced ability to supply nutrients to the cells and to drain waste products can lead to reduced cellular activity and metabolism, causing further degradation of the ECM, while accumulating high levels of toxins that can irritate nerves and cause pain.
As the IVD degeneration progresses, the toxin levels of cytokines and MMPs present in the nucleus pulposus begin to degrade the ECM. In particular, MMPs (mediated by cytokines) begin to cleave the water-retaining portion of proteoglycans, thereby reducing their water-retaining capacity. This degradation results in a reduction in the flexibility of the nucleus pulposus, altering the loading pattern within the intervertebral disc, which in turn can cause the annulus to delaminate. These changes cause greater mechanical instability, which can cause cells to release even more cytokines, leading to MMP upregulation. As this cascade of destruction continues and the IVD degeneration progresses, the disc begins to bulge ("disc herniation") and then eventually ruptures, allowing the nucleus pulposus to contact the spinal cord and cause pain.
Currently, the primary therapy for IVD degeneration is surgical intervention during which the degenerative disc is removed or fused with an adjacent disc. Surgical therapy aims to relieve pain or other symptoms of IVD degeneration, but does not allow for the repair or regeneration of diseased IVD. One method of treating degenerating IVD cells and tissues is to employ cell-based therapies in which diseased tissues are repaired, replaced, and/or remodeled by administration of living cells. Several recent studies have explored the use of cell-based therapies for the treatment of degenerative IVD disorders. For example, U.S. patent nos. 6,352,557 ("Ferree") and 6,340,369 ("FerreeII") teach the harvesting of live IVD cells from a patient, culturing the cells and transplanting them into an injured IVD. Similarly, Alini, Eur. spine J.,2002:11(Supp.2): S215-220(Alini, J. European spinal journal, 2002, Vol. 11 (suppl. 2), p. S215-220) describes the isolation and culture of cells from the nucleus pulposus, the implantation of the cells into a biological matrix, and the injection of the implanted cells into a patient to restore function to an impaired IVD. These approaches, while promising, have shown limited efficacy in repairing degenerative IVD and suffer from complications arising from immune incompatibility between the cell donor and recipient.
An alternative approach to cell-based therapy is the use of stem cells that are capable of dividing and differentiating into the cells that make up the diseased tissue. Stem cell transplantation can be used as a clinical tool to reconstitute target tissues to restore physiological and structural function. The applications of stem cell technology are very wide ranging and include tissue engineering, gene therapy Delivery and cell therapy, i.e., Delivery of biological therapeutic agents to a target site via exogenously supplied living cells or cell components that produce or contain these agents (for a related review, see Tresco, p.a. et al, Advanced Drug Delivery Reviews, 2000; 42:2-37(Tresco, p.a. et al, Advanced Drug Delivery Reviews,2000, volume 42, pages 2-37)). The identification of stem cells has facilitated research aimed at the selective generation of specific cell types for regenerative medicine. One obstacle to achieving the therapeutic potential of stem cell technology is the difficulty in obtaining sufficient numbers of stem cells. Embryonic or fetal tissue is a source of stem cells. Embryonic stem and progenitor cells have been isolated from a variety of mammalian species, including humans, and several such cell types have been demonstrated to be capable of self-renewal and expansion and differentiation into a variety of different cell lineages. However, the availability of stem cells from embryonic and fetal sources raises a number of ethical and ethical issues that have prevented further development of embryonic stem cell therapies.
There is therefore a need in the art for stem cell-based therapies that circumvent the problems associated with embryonic and fetal stem cells. Postpartum tissues such as the umbilical cord and placenta have received attention as alternative sources of pluripotent or multipotent stem cells. For example, methods for recovering stem cells by perfusing the placenta or collecting from umbilical cord blood or tissue have been described. The limitations of obtaining stem cells from these methods are that a sufficient volume of cord blood or a sufficient number of cells cannot be obtained, and that the cell populations obtained from these sources are heterogeneous or have not been characterized.
Thus, a reliable, well characterized, and abundant source of substantially homogeneous stem cell populations capable of differentiating into cells with a phenotype similar to that of endogenous IVD cells would be advantageous for a variety of diagnostic and therapeutic applications to repair, regenerate, and/or replace IVD cells, and to reconstitute and/or remodel IVD tissue.
Disclosure of Invention
In one aspect, provided herein is a method for treating a disease or disorder associated with IVD degeneration (IDD). The method comprises administering an amount of umbilical cord tissue-derived cells effective to treat the disease or disorder.
In another aspect, a pharmaceutical composition for treating a disease or disorder associated with IVD degeneration is provided, the composition comprising a pharmaceutically acceptable carrier and umbilical cord tissue-derived cells in an amount effective to treat the disease or disorder, wherein the umbilical cord tissue from which the cells are obtained is substantially free of blood, and wherein the cells are capable of self-renewal and expansion in culture and have the potential to differentiate into, for example, an IVD cell phenotype, require L-valine for growth, can grow in at least about 5% oxygen, do not produce CD117 or HLA-DR or telomerase, express α smooth muscle actin, and express increased levels of interleukin 8 and reticulon 1 relative to human fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells.
According to another aspect, there is provided a kit for treating a patient having a disease or disorder associated with IVD degeneration, the kit comprising instructions for using the kit in a method of treating a disease or disorder associated with IVD degeneration, a pharmaceutically acceptable carrier, and umbilical cord tissue-derived cells in an amount effective to treat the disease or disorder, wherein the umbilical cord tissue from which the cells are obtained is substantially free of blood, and wherein the cells are capable of self-renewal and expansion in culture and have the potential to differentiate into, for example, an IVD cell phenotype, require L-valine for growth, can grow in at least about 5% oxygen, do not produce CD117 or HLA-DR or telomerase, express α smooth muscle actin, and express elevated levels of interleukin 8 and plasmacytoin 1 relative to human fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells.
In various embodiments, the umbilical cord tissue-derived cells used in the methods, compositions, and/or kits described herein express oxidized low density lipoprotein receptor 1, plasma membrane protein, chemokine receptor ligand 3, and/or granulocyte chemotactic protein 2. In some embodiments, the umbilical cord tissue-derived cells described herein express CD10, CD13, CD44, CD73, and CD 90. In some embodiments, the umbilical cord tissue-derived cells described herein are capable of differentiating into annulus fibrosus and/or nucleus pulposus cells.
In various embodiments, the disease or disorder associated with the degeneration of IVD can be caused or caused by age, trauma, autoimmunity, inflammatory responses, genetic defects, immune complex deposits, and/or combinations thereof. The IVD for targeted therapy can be intact or at any stage of injury or degeneration. For example, the IVD of targeted therapy may be prominent, ruptured, stratified, and/or otherwise damaged or degenerated.
In some embodiments, the methods provided herein comprise administering undifferentiated umbilical cord tissue-derived cells or cell derivatives. Human umbilical cord tissue-derived cells produce beneficial trophic factors including, but not limited to, cytokines, growth factors, protease inhibitors, extracellular matrix proteins that promote survival, growth, and differentiation of endogenous IVD progenitor or precursor cells. The trophic factors described herein may be secreted directly into the host environment by the transplanted human umbilical cord tissue-derived cells. Trophic factors or other cell derivatives can be collected from human umbilical cord tissue-derived cells in vitro and subsequently introduced into a patient.
In some embodiments, the umbilical cord tissue-derived cells described herein are induced in vitro to differentiate into cells of a chondrocyte lineage, and/or into cells exhibiting a phenotype of an annulus fibrosus cell, a nucleus pulposus cell, and/or another IVD-like cell, before, after, or concurrently with cell administration. Thus, in some embodiments, the methods provided herein further comprise the step of inducing the umbilical cord tissue-derived cells to at least partially differentiate in vitro.
In some embodiments, the umbilical cord tissue-derived cells may be genetically engineered to express a gene product, such as, but not limited to, a gene product that facilitates IVD tissue repair and/or regeneration. For example, in some embodiments, the umbilical cord tissue-derived cells are genetically engineered to express trophic factors or other gene products. In some embodiments, the gene product has a trophic effect or otherwise modulates umbilical cord tissue-derived cells, additional cell types administered with umbilical cord tissue-derived cells, endogenous IVD cells, and/or other endogenous cells. In some embodiments, the gene product is a component of an extracellular matrix or an agent that modulates an extracellular matrix. In some embodiments, the gene product stimulates the expression of one or more extracellular matrix proteins.
In some embodiments, the umbilical cord tissue-derived cells are administered with at least one other cell type, such as, but not limited to, an annulus fibrosus cell, a nucleus pulposus cell, a fibroblast, a chondrocyte, a mesenchymal stem cell, an adipose tissue-derived cell, or another pluripotent or multipotent stem cell type. The at least one other cell type can be administered simultaneously with, prior to, or after the umbilical cord tissue-derived cells are administered.
In various embodiments, the at least one agent has a trophic effect on or otherwise modulates the umbilical cord tissue-derived cells, one or more additional cell types administered with the umbilical cord tissue-derived cells, endogenous IVD cells, and/or other endogenous cells.
In various aspects, the administration cells can be administered, targeted, or formulated by injection into the IVD, including, for example, the nucleus pulposus and/or annulus fibrosus of a degenerated IVD. In some embodiments, the cells are administered, administered directionally, or administered formulated such that the cells are encapsulated within an implantable device, or administered by implantation of a device or matrix comprising the cells.
Another embodiment of the invention is a method of treating a disease or disorder associated with degeneration of the intervertebral disc, the method comprising administering to the intervertebral disc a pharmaceutical composition in an amount effective to treat the disease or disorder, the pharmaceutical composition comprising at least (1) a hydrogel and (2) an isolated homogenous population of cells obtained from human umbilical cord tissue, wherein the umbilical cord tissue is substantially free of blood, and wherein the isolated homogenous population of cells is capable of self-renewal and expansion in culture and has the potential to differentiate, but does not express CD117 and/or telomerase the isolated homogenous population of cells may further have any of the following properties (a) express serosal protein, chemokine receptor ligand 3 and granulocyte chemotactic protein, (b) do not produce CD31, CD34 and HLA-DR, (c) express elevated levels of interleukin 8 and serosal protein 1 relative to human fibroblasts, mesenchymal stem cells or iliac crest bone marrow cells, and (d) express CD10, CD13, CD44, CD 2 and CD 90. in another embodiment of the invention, the isolated homogenous population of cells may be administered in vitro by administering at least one of the composition comprising at least one of cells expressing CD13, CD 2, CD1, TGF-CD 3 and/or TGF-CD 3, and/or TGF-CD-c.
Another embodiment of the invention is a method of treating a disease or disorder associated with degeneration of an intervertebral disc, the method comprising administering to an intervertebral disc a hydrogel and an isolated homogenous population of cells obtained from human umbilical cord tissue in an amount effective to treat the disease or disorder, wherein the umbilical cord tissue is substantially free of blood, and wherein the isolated homogenous population of cells is capable of self-renewal, expansion in culture, and has the potential to differentiate, but does not express CD117 and/or telomerase. The isolated homogenous population of cells may also have any of the following characteristics: (a) express plasma membrane protein, chemokine receptor ligand 3, and granulocyte chemotactic protein; (b) does not produce CD31, CD34 and HLA-DR; (c) expresses increased levels of interleukin 8 and reticulon 1 relative to human fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells; and (d) expresses CD10, CD13, CD44, CD73, and CD 90. In another embodiment, the isolated homogenous population of cells expresses oxidized low density lipoprotein receptor 1, plasma membrane protein, chemokine receptor ligand 3, and granulocyte chemotactic protein. The hydrogel and the isolated homogenous cell population may be administered by injection. The hydrogel can be administered simultaneously with, or before or after administration of the isolated homogenous population of cells obtained from human umbilical cord tissue. In one embodiment, the isolated homogenous population of cells is administered within an implantable device. In another embodiment, the method further comprises administering at least one additional cell type simultaneously with, or prior to, or subsequent to, administering the isolated homogenous population of cells obtained from human umbilical cord tissue. The at least one other cell type can be engineered to express at least one exogenous gene product (e.g., a trophic factor or an exogenous gene product that modulates the expression of one or more extracellular matrix proteins).
The methods may further include administering at least one agent, such as trophic factors (e.g., TGF- β, GDF-5, PDGF-BB, and TIMP1) that may have a trophic effect on the isolated homogenous cell population obtained from human umbilical cord tissue.
Another embodiment of the invention is a method of treating a disease or condition associated with degeneration of an intervertebral disc comprising administering an amount of hydrogel effective to treat the disease or condition. Any hydrogel known in the art, such as those disclosed herein, may be used in this method. In one embodiment, the hydrogel comprises fibrinogen and thrombin. In another embodiment, the hydrogel comprises a fibrin glue such as
The foregoing and other features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings.
Drawings
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, the drawings show embodiments of the invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples and instrumentalities shown.
Figures 1 and 2 show lumbar MRI (see example 12). The median sagittal plane T2 weighted MRI showed a healthy looking disc in the control group. The intervertebral discs in the puncture set degenerated (darkened and lost height). The degeneration of the discs in the treated group was not as pronounced as the discs in the punctured group. Specifically, figures 1 and 2 show sample T2-weighted median sagittal plane lumbar MRI images of L1-2 through L5-6 at time points 0 (before circular puncture), 3 weeks (before injection surgery), 6 weeks, and 12 weeks (before sacrifice). The punctured discs (L2-3, L3-4 and L4-5) are delineated by boxes (L2-3, L3-4 and L4-5 from top to bottom). The unpunctured control discs (L1-2 and L5-6) did not show any signs of degeneration. As expected, the intervertebral disc of the control sample did not degenerate (fig. 1A). The punctured disc (fig. 1B) became smaller and darker over time, indicating that degeneration had occurred. The discs treated with vehicle (fig. 1C), cell + buffer (fig. 2A) and cell + vehicle (fig. 2B) after puncture demonstrated less pronounced degeneration over time than the punctured discs (fig. 1B).
Figure 3 shows T2 weighted MRI (disc volume and MRI index) (see example 12). Specifically, combining L2-3, L3-4, and L4-5 (treated discs), mean NP MRI area expressed as a percentage of time 0 values (fig. 3A), and MRI index (fig. 3B) for each subgroup of rabbits, confirms that the largest area and index reduction occurred with the punctured group over time, while the smaller area and index reduction occurred with the group treated with vehicle, cells in buffer (B + C), or cells in vehicle (C + C) after puncturing. In the context of figure 3 of the drawings,
MRI index NP area signal intensity
FIG. 4 shows the average normalized total displacement (ramp phase + creep) curve of the embedded L3-4FSU after 12 weeks (see example 12). The time-dependent displacement under constant load produces a creep curve that appears different. Buffer + cells behaved more like the puncture group, vehicle + cells behaved more like the control, vehicle alone somewhere in between. The average creep curve for each case was generated. The axial test produces a creep curve that appears different early in the test (time 0 to 200 seconds). The dashed border represents the measurement standard deviation. The curves generated by the spiked groups and buffer + cells appeared similar, as did the curves for the control and vehicle + cells. Each of these groups appeared to be different from the curve generated by the vector group.
Figures 5,6 and 7 show histological sagittal sections of disc L4-5 obtained after sacrifice at 12 weeks for each treatment group, stained with hematoxylin-eosin and magnified 20-fold and 100-fold (see example 12). FIG. 5A shows a histological sagittal section of the disc L4-5 of the control group. Fig. 5B shows a histological sagittal section of the puncture set of intervertebral disc L4-5. Fig. 6A shows a histological sagittal section of the disc L4-5 of the vehicle set. FIG. 6B shows a histological sagittal section of the disc L4-5 with buffer and cell sets. Figure 7 shows a histological sagittal section of the disc L4-5 of the vector and cell set.
Detailed Description
Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be understood in a manner consistent with the definitions provided herein.
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.
As used herein, the term "about," when referring to a measurable value such as an amount, time interval, etc., is meant to encompass variations from the specified value of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1%, as such variations are suitable for performing the disclosed methods.
"derived" is used to indicate that the cells are obtained from their biological source and grown, expanded in culture, immortalized or otherwise manipulated in vitro.
"isolated" means "altered in nature by manual manipulation". If a molecule or composition is naturally occurring, it has been "isolated" when it has been altered or removed from its original environment or both.
The terms "expressed," "expressed," or "expression" of a nucleic acid molecule or gene refer to the biosynthesis of a gene product, e.g., a polypeptide.
A "trophic factor" is a substance that promotes cell survival, growth, differentiation, proliferation, and/or maturation or stimulates an increase in the biological activity of a cell. Cell derivatives refer to any material that can be obtained from cells and include cell conditioning media, cell lysates, extracellular matrix proteins, trophic factors, cellular components, cellular membranes.
By "degeneration" is meant any physical injury, degeneration or trauma to the IVD.
"lesion" refers to any structural or functional indicator of deviation from the normal state of a cell, tissue, organ or system, as measured by any method suitable in the art.
A "disease" is any deviation from or impairment of the health, state or function of a cell, tissue, organ, system or organism in general, as measured by any method suitable in the art.
"treated," "treated," or "treatment" refers to any indication of success or success in reducing or ameliorating a disease, injury, or condition, including any objective or subjective parameter such as reduction of symptoms, alleviation, attenuation, or making a patient more tolerant to the disease, injury, or condition, slowing the rate of degeneration or decline, reducing the extent of degeneration end point failure, or improving the physical or mental well-being of the subject. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including results of physical examination, neurological examination, and/or mental identification.
An "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, material, or composition as described herein effective to achieve a particular biological result, such as, but not limited to, the biological results disclosed, described, and recited herein. Such results may include, but are not limited to, treatment of IVD disease or injury in a subject, as determined by any method suitable in the art.
By "pharmaceutically acceptable" is meant those properties and/or substances that are acceptable to the patient from a pharmacological/toxicological perspective and to the pharmaceutical chemist from a physical/chemical perspective related to composition, formulation, stability, patient acceptance and bioavailability. By "pharmaceutically acceptable carrier" is meant a medium that does not affect the efficacy of the biological activity of the active ingredient and is non-toxic to the host to which it is administered.
It has been found that diseases and disorders associated with degeneration of intervertebral disc (IVD) can be treated by administering umbilical cord tissue-derived cells as described herein. Advantageously, the methods, compositions, and kits provided herein facilitate repair and regeneration of a degenerated IVD, thereby alleviating one or more symptoms associated with the degeneration of IVD. Thus, in one aspect, there is provided a method for treating a disease or disorder associated with the degeneration of IVD, the method comprising administering umbilical cord tissue-derived cells to the IVD in an amount sufficient to treat the disease or disorder.
In various embodiments, the disease or disorder associated with the degeneration of IVD can be caused or caused by age, trauma, autoimmunity, inflammatory responses, genetic defects, deposition of immune complexes (e.g., formation of scar tissue), and/or combinations thereof. The IVD for targeted therapy can be intact or at any stage of injury or degeneration. For example, the IVD of targeted therapy may be prominent (e.g., where a portion of the annulus fibrosus has a bulge or other protrusion), disrupted (e.g., where at least a portion of the annulus fibrosus is disrupted, resulting in a decrease in pressure and/or volume of the nucleus pulposus), delaminated (e.g., where two or more layers of the annulus fibrosus have separated), and/or otherwise damaged or degenerated (e.g., where the annulus fibrosus has a crack, tear, etc., and/or where the extracellular matrix is degraded or altered).
In various embodiments, umbilical cord tissue-derived cells are administered to a degenerated IVD, for example, by injection, transplantation, implantation, injection, provided as a matrix-cell complex, or any other means known in the art for providing cell therapy. In some embodiments, the cells are administered directly to the annulus fibrosus and/or nucleus pulposus of the IVD. In some embodiments, the cells are administered indirectly to the IVD. For example, cells can be in an aqueous carrier, encapsulated in a device, or seeded in a matrix, which is then implanted in or near a degenerated IVD. Aqueous carriers include, but are not limited to, physiological buffered solutions such as buffered saline, phosphate buffered saline, Hank's balanced saline, Tris buffered saline, and Hepes buffered saline. In various embodiments, the device, matrix or other cell reservoir may be implanted such that it is attached to the outer wall of the annulus fibrosus, or at a location outside but adjacent to the wall of the annulus fibrosus, or adjacent to the endplates of the vertebral body surrounding the IVD.
In some embodiments, the cells are administered in the form of a device, such as a matrix-cell complex. Device materials include, but are not limited to, bioabsorbable materials such as collagen, 35/65 poly (epsilon-caprolactone) (PCL)/polyglycolic acid (PGA), PANACRYLTMBioabsorbable construct, VicrylTMThe polysaccharide lactic acid 910, as well as self-assembling peptides and non-absorbent materials such as fluoropolymers (e.g.,
In one embodiment, the umbilical cord tissue-derived cells are administered to the degenerated IVD as part of a composition comprising a hydrogel. In another embodiment, umbilical cord tissue-derived cells are administered to the degenerated IVD along with a hydrogel.
The cells described herein can also be seeded onto a three-dimensional matrix, such as a scaffold implanted in vivo, where the seeded cells can proliferate on or in the framework, or help establish replacement tissue in vivo with or without cooperation with other cells. Growth of the umbilical cord tissue-derived cells on the three-dimensional framework preferably results in the formation of a three-dimensional tissue or architecture thereof, which can be utilized in vivo, for example, to repair and/or regenerate damaged or diseased tissue.
Cells can be seeded onto a three-dimensional framework or matrix, such as a scaffold, foam, electrospun scaffold, non-woven scaffold, porous or non-porous microparticle, or hydrogel, and administered accordingly. The frame may be configured in a variety of shapes, such as substantially flat, substantially cylindrical, or tubular, or may be a completely free form as may be required or desired for the orthotic structure in question. Two or more substantially flat frames may be placed on top of one another and secured together as desired to create a multi-layered frame.
On such three-dimensional frameworks, cells can be co-administered with other cell types, or other soft tissue type progenitor cells (including stem cells). When grown in a three-dimensional system, the proliferating cells can mature and properly segregate to form components of adult tissue that resemble corresponding tissue that naturally occurs in vivo.
The matrices described and recited herein can be designed such that the matrix structure supports umbilical cord tissue-derived cells and does not subsequently degrade, supports cells from seeding until the time the host tissue remodels the tissue graft, or allows seeded cells to attach, proliferate, and develop into a tissue structure having sufficient mechanical integrity to support themselves ex vivo, at which time the matrix degrades.
The matrices, scaffolds such as foamed nonwovens, electrospun structures, microparticles and self-assembling systems contemplated for use herein may be combined with any one or more cells, growth factors, drugs or other components, such as bioactive agents that promote tissue healing, regeneration, repair or ingrowth, or stimulate angiogenesis or innervation thereof or otherwise enhance or improve the therapeutic effect or practice of the invention, and implanted together with the cells of the invention.
In some embodiments, the cells described herein can grow freely in culture, be removed from culture, and seeded onto a three-dimensional framework. At a cell concentration, e.g. about 10 per ml6To 5X 107Seeding the three-dimensional framework with individual cells preferably allows the three-dimensional support material to be established in a relatively short period of time. Furthermore, in some applications, it may be preferable to use a greater or lesser number of cells depending on the desired result.
In some aspects, it may be useful to reconstitute the cellular microenvironment present in vivo in culture, such that the extent of cell growth prior to implantation in vivo or use in vitro may vary. The cells may be seeded onto the frame before or after forming into the desired shape for implantation, e.g., rope, tube, filament, etc. After seeding the cells onto the frame, the frame is preferably incubated in a suitable growth medium. During incubation, the seeded cells will grow and coat the framework, and may, for example, bridge or partially bridge any interstitial spaces therein. It is preferred, but not necessary, to grow the cells to an appropriate extent that reflects the in vivo cell density of the tissue being repaired or regenerated. In other embodiments, the presence of cells (even in small numbers) on the framework promotes endogenous healthy cell ingrowth, facilitating, for example, healing of damaged or injured cells.
Examples of substrates, such as scaffolds, that may be used in various aspects of the invention include pads, porous or semi-porous foams, self-assembling peptides, and the like. The nonwoven mat may be formed, for example, using fibers composed of natural or synthetic polymers. In a preferred embodiment, the trade name ViCRYL is usedTMAbsorbable copolymers of glycolic and lactic acids (PGA/PLA) sold by erickang corporation of sameville, new jersey (Ethicon, inc., Somerville, NJ) form pads. By as described in U.S. Pat. No.6,355,699Foams composed of, for example, poly (epsilon-caprolactone)/poly (glycolic acid) (PCL/PGA) copolymers, formed by the methods discussed, for example, by freeze-drying or lyophilization, can also be used as scaffolds.
The gel also forms a suitable matrix as used herein. Examples of gels include injectable gels, in situ polymerizable gels, and hydrogels; the gel may be composed of self-assembling peptides. These materials are generally used as support materials for tissue growth. For example, when used as an injectable gel, the gel may be composed of water, saline solution or physiological saline solution and a gelling material. Gelling materials include, but are not limited to, proteins such as collagen, elastin, thrombin, fibronectin, gelatin, fibrin, tropoelastin, polypeptides, laminin, proteoglycans, fibrin glues, fibrin clots, Platelet Rich Plasma (PRP) clots, Platelet Poor Plasma (PPP) clots, self-assembling peptide hydrogels, and atelocollagen; polysaccharides such as pectin, cellulose, oxidized cellulose, chitin, chitosan, agarose, hyaluronic acid; polynucleotides such as ribonucleic acids, deoxyribonucleic acids, and others such as alginates, cross-linked alginates, poly (N-isopropylacrylamide), poly (alkylene oxides), copolymers of poly (ethylene oxide) -poly (propylene oxide), poly (vinyl alcohol), polyacrylates, glyceryl monostearate-co-succinic acid/polyethylene glycol (MGSA/PEG) copolymers, and combinations thereof. In one embodiment, the gelling material (i.e. hydrogel) comprises fibrinogen (factor I), e.g. recombinant fibrinogen or fibrinogen purified from blood. In another embodiment, the hydrogel comprises fibrinogen and thrombin. In another embodiment, the gel is
Generally, hydrogels are crosslinked polymeric materials that can absorb more than 20% of their body weight in water while maintaining a unique three-dimensional structure. In addition, hydrogels have a high permeability to oxygen, nutrients and other water-soluble metabolites. This definition includes dried crosslinked polymers that swell in an aqueous environment as well as water swellable materials. Many hydrophilic polymers can be crosslinked to form hydrogels, whether the polymer is of biological origin, semi-synthetic or fully synthetic. Hydrogels can be made from synthetic polymeric materials. Such synthetic polymers can be specifically designed in terms of a range of properties and predictable lot-to-lot consistency, and represent a reliable source of material that is generally free of immunogenicity concerns. In one embodiment of the invention, the hydrogel is formed from self-assembling peptides, such as those discussed in U.S. Pat. Nos. 5,670,483 and 5,955,343, U.S. patent publication No.2002/0160471, PCT patent application No. WO02/062969, the disclosures of which are incorporated herein by reference in their entirety.
Properties that make hydrogels particularly valuable as matrices for the present invention include equilibrium swelling capacity, adsorption kinetics, solute permeability, and their in vivo performance characteristics. The permeability to a compound depends in part on the degree of swelling or water content and the rate of biodegradation. Since the mechanical strength of the gel decreases in proportion to the degree of swelling, it is also within the contemplation of the invention that the hydrogel may be attached to the substrate, and thus the composite system enhances mechanical strength. In alternative embodiments, the hydrogel may be impregnated within a porous substrate to simultaneously achieve the mechanical strength of the substrate and the useful delivery properties of the hydrogel.
Degradable networks formed in situ are also suitable for use in the present invention (see, e.g., Anseth, K.S. et al, J.controlled Release, 2002; 78: 199-. These materials can be formulated into a fluid suitable for injection, which can then be induced to form a degradable hydrogel network in situ or in vivo by a variety of means (e.g., changing temperature, pH, exposure to light).
In some embodiments, the frame may be a felt, which may be constructed of multifilament yarns made of bioabsorbable materials, such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarns are formed into a felt using standard textile processing techniques including crimping, cutting, carding and needling. The cells of the invention may be seeded onto a foam scaffold, which may be a composite structure. Furthermore, the three-dimensional frame may be molded into a useful shape, such as a particular structure in or near the IVD to be repaired, replaced, or enhanced.
The frame may be treated prior to seeding with cells of the invention to improve cell adhesion. For example, prior to seeding the cells of the invention, a nylon matrix may be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon. Polystyrene can be similarly treated with sulfuric acid.
In addition, the outer surface of the three-dimensional framework may be modified to improve cell adhesion or growth and tissue differentiation, for example, by plasma coating the framework or adding one or more proteins (e.g., collagen, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate), cell matrices, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, among others.
The stent is constructed of or treated with a material that renders it non-thrombogenic. These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of such materials and treatments include, but are not limited to, natural materials such as basement membrane proteins such as laminin and type IV collagen, synthetic materials such as ePTFE, and blocked polyurethaneurea silicones such as
For example, different proportions of the various types of collagen deposited on the framework may affect the growth of tissue-specific cells or other cells that may be subsequently seeded on the framework or may be grown on the in vivo structure. Alternatively, the frame may be seeded with a mixture of cells that synthesize the appropriate collagen type required. Depending on the tissue to be cultured, the appropriate type of collagen to be seeded onto the frame or produced by the cells seeded thereon may be selected. For example, the relative amounts of collagen fibers and elastic fibers present in the framework can be adjusted by controlling the ratio of collagen-producing cells to elastin-producing cells in the initial inoculum.
The seeded or injected three-dimensional framework of the invention may be used to transplant or implant cultured cells obtained from the matrix or the matrix itself cultured in vivo. Three-dimensional scaffolds may be used according to the present invention to replace or augment existing tissue, to introduce new or altered tissue, to remodel artificial prostheses, or to join biological tissues or structures together.
In some embodiments, the cells can be administered (e.g., injected) into the IVD through a needle, such as a small gauge needle. In some embodiments, the needle has a gauge of about 22 gauge or less to reduce the likelihood of IVD herniation. When injecting a volume into the nucleus pulposus, it is desirable that the volume of drug delivered is no more than about 3ml, preferably no more than about 1ml, more preferably between about 0.1 and about 0.5 ml. When injected in these smaller amounts, it is believed that the added volume does not cause a significant pressure increase in the nucleus pulposus. If the volume of the direct injection of the formulation is high enough to cause concern about over-pressurization of the nucleus pulposus, it is preferred that at least a portion of the nucleus pulposus be removed prior to the direct injection. In some embodiments, the volume of the nucleus pulposus removed is substantially similar to the volume of formulation to be injected. For example, the volume of the nucleus pulposus removed may be within about 80-120% of the volume of formulation to be injected. In some embodiments, the umbilical cord tissue-derived cells are concentrated prior to administration.
Cells useful in the methods, compositions, and kits provided herein can be derived from mammalian umbilical cord that can be recovered immediately or shortly after term or early termination of pregnancy, e.g., after discharge after birth or surgical removal post-cesarean section. Prior to cell isolation, blood and debris are removed from the umbilical cord tissue, for example, by washing with any suitable medium or buffer.
The cells may be isolated from the umbilical cord tissue by mechanical force or by enzymatic digestion. Preferred enzymes are metalloproteases, neutral proteases and mucolytic proteases. For example, various combinations of collagenase, dispase, and hyaluronidase can be used to dissociate cells from umbilical cord tissue. The skilled artisan will recognize that a variety of such enzymatic treatments are known in the art for isolating cells from various tissue sources. For example,
The umbilical cord tissue-derived CELLs are cultured in any medium capable of sustaining CELL growth, such as, but not limited to, DMEM (high or low glucose), high grade DMEM, MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Hayflick's medium, Iscove's modified Dulbecco's medium, Mesenchymal Stem CELL Growth Medium (MSCGM), DMEM/F12, RPMI 1640, and GRO-FREE, medium may be supplemented with one or more components including, for example, fetal bovine serum, preferably about 2-15% (by volume) bovine serum albumin, PDGF/EGF), bovine serum-derived growth factor (VEGF), bovine serum-derived factor (EGF), bovine serum-derived growth factor (VEGF), bovine serum-derived factor (EGF), bovine serum-2-15% (by volume), bovine serum-derived factor (VEGF), bovine serum-derived factor (EGF), bovine serum-derived factor (VEGF), bovine serum-derived factor (VEGF), bovine serum factor-derived factor-2), bovine serum-derived factor (bovine serum-derived factor-derived), bovine serum factor-derived factor (bovine serum factor-derived), bovine serum factor-derived), bovine serum factor (bovine serum factor-derived), bovine serum factor-derived from one or bovine serum factor (bovine serum factor-derived), bovine serum factor-derived from human serum factor-derived from animal origin (bovine serum factor-derived).
Cells were seeded in culture flasks at a density that allowed for cell growth. In one embodiment, the cells are filled with about 0 to about 5 volume percent CO2And (5) culturing. In some embodiments, the cells are about 2 to about 25% O in space gas2Below, it is preferred that O be present in an amount of about 5 to about 20% of the air2And (5) culturing. The cells are preferably cultured at about 25 to about 40 ℃, more preferably at 37 ℃. The medium in the flask may be static or may be agitated, for example, with a bioreactor. The umbilical cord tissue-derived cells are preferably grown under low oxidative stress (e.g., glutathione, vitamin C, catalase, vitamin E, N-acetylcysteine added), meaning that there is no or little free radical damage to the cultured cells.
The umbilical cord tissue-derived cells can be passaged or removed into separate culture flasks containing fresh medium of the same or different type as the initially used medium, and in which the cell population can be mitotically expanded. The cells of the invention may be used at any time point between passage 0 and senescence. The cells are preferably passaged from about 3 to about 25 times, more preferably passaged from about 4 to about 12 times, and preferably passaged 10 or 11 times. Cloning and/or subcloning may be performed to confirm that the clonal population of cells has been isolated.
In one embodiment of the invention, the cells may be grown (expanded) on microcarriers. Microcarriers are particles, beads or globules that can be used for the attachment and growth of anchorage-dependent cells in culture. Microcarriers can be solid, porous, or solid cores with porous coatings. Exemplary suitable microcarriers include, but are not limited to, CytodexCytodex
The different cell types present in the umbilical cord tissue can be separated into subpopulations. This can be accomplished using standard techniques for cell isolation including, but not limited to, enzymatic treatment; cloning and selection of specific cell types, such as, but not limited to, selection based on morphological and/or biochemical markers; selective growth of desired cells (positive selection), selective destruction of undesired cells (negative selection); separation based on differences in cell agglutination in the mixed population as derived, for example, using soybean agglutinin; freezing and thawing; differential adhesion of cells in a mixed population; filtering; conventional and zonal centrifugation; centrifugal elutriation (countercurrent centrifugation); separating by unit gravity; counter-current distribution; electrophoresis; fluorescence Activated Cell Sorting (FACS); and so on.
Examples of cells isolated from umbilical cord tissue were deposited at the American Type Culture Collection (American Type Culture Collection) on day 10, 6.2004 and assigned ATCC accession numbers as follows: (1) strain name UMB022803(P7) was designated as accession number PTA-6067; and (2) strain name UMB022803 (P17) is designated as accession number PTA-6068.
Umbilical cord tissue-derived cells can be characterized, for example, by: growth characteristics (e.g., population doubling capacity, doubling time, number of passages to senescence), karyotyping (e.g., normal karyotype; maternal or neonatal lineage), flow cytometry (e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry (e.g., for detecting epitopes), gene expression profiles (e.g., gene chip arrays; polymerase chain reactions (e.g., reverse transcriptase PCR, real-time PCR and conventional PCR)), protein arrays, protein secretion (e.g., by plasma coagulation assay or analysis of PDC conditioned media, e.g., by enzyme-linked immunosorbent assay (ELISA)), mixed lymphocyte reactions (e.g., as a measure of PBMC stimulation), and/or other methods known in the art.
In various aspects, the umbilical cord tissue-derived cells have one or more of the following growth characteristics: l-valine is required for growth in culture; capable of growing in an atmosphere comprising from about 5% to at least about 20% oxygen; has the potential to undergo at least about 40 doublings before reaching senescence when cultured; and/or attachment and amplification on coated or uncoated tissue culture flasks, wherein the coated tissue culture flasks comprise a coating of gelatin, laminin, collagen, polyornithine, vitronectin, or fibronectin.
In some embodiments, the cells have a normal karyotype that is maintained upon passaging of the cells. Karyotyping is particularly useful for identifying and differentiating neonatal cells from maternal cells derived from the placenta. Methods for karyotyping are available and known to those skilled in the art.
In some embodiments, the cells may be characterized by production of a particular protein, including production of at least one of tissue factor, vimentin, and α -smooth muscle actin, and production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr- α, PD-L2, and HLA-A, B, C cell surface markers, as detected, for example, by flow cytometry.
In some embodiments, the cell has an increased expression of a gene encoding at least one of interleukin 8, reticulon 1, chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, α), chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2), chemokine (C-X-C motif) ligand 3, tumor necrosis factor, α -inducible protein 3, relative to a human cell that is a fibroblast, mesenchymal stem cell, or iliac crest bone marrow cell.
In other embodiments, the cells have reduced expression relative to human cells that are fibroblasts, mesenchymal stem cells or iliac crest bone marrow cells of at least one of the genes encoding for disintegrin, the rat synbox 2, heat shock 27kDa protein 2, chemokine (C-X-C motif) ligand 12 (stromal cell derived factor 1), elastin (aortic stenosis on valve, Williams-Boylen syndrome), human (Homo sapiens) mRNA, cDNA DKFZp586M2022 (from clone DKFZp586M2022), mesenchymal synbox 2 (growth termination specific synbox), sine oculis synbox homolog 1 (Drosophila), crystallin, α B, morphogenetic disorder associated activator 2, ZP586B2420 protein, like excretory nexin 1, tetranectin (plasminogen binding protein), src synbox 3(SH3) and cysteine-rich domain of cyclin, the rat actin-related activator domain, the rat glycoprotein subunit II, the putative protein, the rat glycoprotein subunit II, the rat glycoprotein subunit of the rat glycoprotein family of the rat, the.
In some embodiments, the cells can be characterized by secreting at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1 in some embodiments, the cells can be characterized by not secreting at least one of TGF- β 2, ANG2, PDGFbb, MIP1a, and VEGF, as detected by ELISA.
In some preferred embodiments, the cell has two or more of the growth, protein/surface marker production, gene expression, or substance secretion characteristics listed above. In some embodiments, it is preferred that the cells have three, four, or five or more of the properties. In some embodiments, it is preferred that the cells have six, seven, or eight or more of the properties. In some embodiments, it is preferred that the cells have all of the above properties. In other embodiments, the umbilical cord-derived cells have any of the characteristics disclosed in U.S. patent nos. 7,510,873 and 7,524,489, the disclosures of which are incorporated herein by reference in their entireties.
Preferred cells for use in aspects of the invention include cells having the above properties, more particularly cells wherein the cells have a normal karyotype and proceed with passage, maintain a normal karyotype, and further wherein the cells express each of the markers CD10, CD13, CD44, CD73, CD90, PDGFr- α, and HLA-A, B, C, wherein the cells produce immunologically detectable proteins corresponding to the listed markers.
In some preferred aspects, the methods comprise administering cells obtained or isolated from human umbilical cord tissue to a degenerated IVD, wherein the cells are capable of self-renewal and expansion in culture, require L-valine for growth, can grow in at least about 5% oxygen, do not produce CD117 or HLA-DR or telomerase, express α smooth muscle actin, and express increased levels of interleukin 8 and reticulin 1 relative to human fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells.
Some cells with the potential to differentiate in the direction that produces the various phenotypes are unstable and therefore can differentiate spontaneously. Thus, in some embodiments, cells that do not spontaneously differentiate are preferred. For example, some preferred cells are substantially stable to the cellular markers produced on their surface when cultured in a growth medium, and to the expression pattern of various genes, e.g., as determined by, e.g., a nucleic acid or polypeptide array using gene expression profiling. Such cells remain substantially constant upon passage, after multiple population doublings, for example in terms of their surface marker properties.
In some embodiments, the methods provided herein induce umbilical cord tissue-derived cells to differentiate along the IVD cellular pathway towards an IVD cell phenotype, or progenitor cells, or more primitive related cells as previously described. The umbilical cord tissue-derived cells may be integrated into the IVD of the patient, or may provide support for the growth or stimulation of differentiation of naturally occurring IVD stem cells. The survival of the administered cells is not a determinant of the success or outcome of their use in achieving an improvement in the disease or disorder associated with IVD degeneration and/or the overall health of the patient. In some embodiments, the cells preferably integrate, propagate, or survive at least partially in the patient. In some embodiments, the patient benefits from the treatment, for example, from the ability of the cells to support the growth of other cells (including stem or progenitor cells present in the IVD and/or surrounding tissue), from tissue ingrowth or vascularization of the tissue, and/or from the presence of beneficial cytokines, chemokines, cytokines, and the like, but without the cells integrating or proliferating in the patient. In some aspects, the patient benefits from therapeutic treatment with the cells, but the cells do not survive long term in the patient. For example, in some embodiments, the number, viability, or biochemical activity of the cells gradually decreases. In some embodiments, such a decrease may be preceded by an active phase, such as growth, division, or biochemical activity. In some embodiments, aged, non-viable, or even dead cells can have a beneficial therapeutic effect.
In some aspects, the methods of the invention may further comprise assessing an improvement in IVD structure and/or function, and/or an improvement in overall health, of the patient. Such assessment may be made according to any method suitable in the art, including those described and recited herein.
In some embodiments, the umbilical cord tissue-derived cells may be administered in conjunction with one or more other cell types, including but not limited to other pluripotent or multipotent cells, or chondrocytes, chondroblasts, osteocytes, osteoblasts, osteoclasts, osteocytes, or bone marrow cells. The different types of cells can be mixed with the umbilical cord tissue-derived cells immediately or shortly before administration, or they can be co-cultured together for a period of time before administration. In some embodiments, the umbilical cord tissue-derived cell population supports survival, proliferation, growth, maintenance, maturation, differentiation, and/or increased activity of one or more other cell types administered in conjunction with the umbilical cord tissue-derived cells, and/or vice versa.
In some embodiments, the umbilical cord tissue-derived cells provide nutritional support to other cell types administered therewith, and/or vice versa. In some embodiments, it is desirable that the umbilical cord tissue-derived cells are contacted with co-cultured cells. This can be achieved, for example, by seeding the cells as a heterogeneous cell population into a culture medium or onto a suitable culture substrate. Alternatively, the umbilical cord tissue-derived cells may be grown first to confluence and then used as a substrate for co-culture of the cells. In other embodiments, the co-cultured cells can be cultured by contacting the co-cultured cells with conditioned medium of umbilical cord tissue-derived cells, extracellular matrix, and/or cell lysate.
In various embodiments, the umbilical cord tissue-derived cells can be administered in conjunction with a bioactive agent, such as an agent that modulates proliferation, differentiation, and/or other cellular activity. The agent may be administered before, after, or simultaneously with the administration of the umbilical cord tissue-derived cells. The particular agent selected may be determined by a medical professional directing the treatment of the patient and may vary according to the particular needs or conditions of the patient. The selected agent may be used for various purposes, such as, but not limited to, facilitating cellular administration, improving repair and/or regeneration of an IVD, improving the overall health of a patient, reducing pain, reducing or preventing rejection of transplanted cells, and the like.
Examples of agents that may be administered with the umbilical cord tissue-derived cells include, but are not limited to, vitamins and other nutritional supplements; an antithrombotic agent; an anti-apoptotic agent; an anti-inflammatory agent; immunosuppressive agents (e.g., cyclosporin, rapamycin); an antioxidant; a hormone; a glycoprotein; fibronectin, peptides and proteins; saccharides (monosaccharides and/or complex sugars); proteoglycan; oligonucleotides (sense and/or antisense DNA and/or RNA); bone Morphogenetic Protein (BMP); a differentiation factor; antibodies (e.g., antibodies to infectious agents, tumors, drugs, or hormones); and gene therapy agents. In some embodiments, the agent is a substance that reduces one or more symptoms of a disease or disorder associated with IVD degeneration, such as analgesia, anti-inflammatory, anesthetic, muscle relaxant, or a combination thereof.
In some embodiments, the additional agent is a trophic factor or other agent that has a trophic effect on cells derived from umbilical cord tissue, on endogenous IVD cells (e.g., pericytes, nucleus pulposus cells), and/or on other endogenous cells (e.g., connective tissue progenitor cells). in some embodiments, the trophic factor is a trophic factor secreted by cells derived from umbilical cord tissue, in which case it may be derived from such a population of cells derived from umbilical cord tissue or from another source.examples of such factors or agents include, but are not limited to, members of the fibroblast growth factor family, including acidic and basic fibroblast growth factors (FGF-1 and FGF-2) and FGF-4, members of the platelet-derived growth factor (PDGF) family, including PDGF-AB, PDGF-BB and PDGF-AA, members of the insulin-like growth factor (IGF) family, including IGF-I and IGF-II, the TGF- β superfamily, including GF- β,2 and 3 (including MP-52), the class-inducible factor, the vascular growth factor (CSF), the VEGF) family, the VEGF-2, EGF-2 family, the EGF-7 family, the BMP-EGF family, the BMP-7 family, and BMP-2 family, and BMP-1, and BMP-2 family, and BMP-1 analogs.
In some embodiments, the growth factor is TGF- β, more preferably, TGF- β is administered in an amount between about 10ng/ml and about 5000ng/ml, such as between about 50ng/ml and about 500ng/ml, such as between about 100ng/ml and about 300 ng/ml.
In some embodiments, the platelet concentrate is provided as an additional therapeutic agent. In some embodiments, the platelet concentrate is autologous. In some embodiments, the platelet concentrate is Platelet Rich Plasma (PRP). PRP is advantageous because it contains growth factors that can re-stimulate the growth of ECM, and because its fibrin matrix provides a scaffold for new tissue growth. In some embodiments, the additional agent is a cell lysate, soluble cell components, membrane-rich cell components, cell culture medium (e.g., conditioned medium), or extracellular matrix derived from umbilical cord tissue-derived cells or other cells.
In some embodiments, the umbilical cord tissue-derived cells are administered in combination with an HMG-CoA reductase inhibitor including, but not limited to, simvastatin, pravastatin, lovastatin, fluvastatin, cerivastatin, and atorvastatin.
In some embodiments, the umbilical cord tissue-derived cells are genetically engineered to express one or more agents, such as, but not limited to, one or more additional therapeutic agents described herein. The cells of the invention may be engineered using any of a variety of vectors, including but not limited to, integrative viral vectors, such as retroviral vectors or adeno-associated viral vectors; non-integrative replication vectors, such as papilloma virus vectors, SV40 vectors, adenovirus vectors; or a replication-defective vector. Other methods of introducing DNA into cells include the use of liposomes, electroporation, particle guns, or by direct DNA injection.
The host cell is preferably transformed or transfected with DNA controlled by or operably linked to one or more appropriate expression control elements, e.g., promoter or enhancer sequences, transcription terminators, polyadenylation sites, and the like, and a selectable marker.
After introduction of the exogenous DNA, the engineered cells can be grown in an enrichment medium and then changed to a selective medium. Selectable markers in the exogenous DNA confer resistance to selection, allowing the cells to stably integrate the exogenous DNA, e.g., on a plasmid, into their chromosomes and grow into colonies, which can then be cloned and expanded into cell lines. The method can be advantageously used to engineer cell lines to express gene products.
Any promoter may be used to drive expression of the inserted gene. For example, viral promoters include, but are not limited to, CMV promoter/enhancer, SV40, papilloma virus, EB virus, or elastin gene promoters. Preferably, the control elements used to control the expression of the gene of interest should allow for the regulated expression of the gene such that the product is synthesized only when needed in vivo. If transient expression is desired, it is preferred to use constitutive promoters in non-integrative and/or replication-defective vectors. Alternatively, inducible promoters may be used to drive expression of the inserted gene as necessary. Inducible promoters include, but are not limited to, promoters associated with metallothionein and heat shock proteins.
The cells of the invention may be genetically engineered to "knock out" or "knock down" the expression of factors that contribute to inflammation or rejection at the site of implantation. Negative regulation techniques for reducing the expression level of a target gene or the activity level of a target gene product will be discussed below. As used herein, "down-regulation" refers to a decrease in the level and/or activity of a target gene product relative to the level and/or activity of the target gene product when no regulatory treatment is performed. Expression of a gene can be reduced or knocked out using a variety of techniques, including, for example, suppression of expression using homologous recombination techniques to completely inactivate the gene (often referred to as "knock out"). Typically, a positive selection marker (e.g., neo) is inserted into an exon encoding a region of interest in a protein (or an exon at the 5' end of that region) to prevent the production of normal mRNA from the target gene and cause gene inactivation. Genes can also be inactivated by introducing a deletion in a portion of the gene, or by deleting the entire gene. By using a construct having two regions homologous to the target gene that are located far apart in the genome, the sequence between the two regions can be deleted (Mombaerts et al, Proc. Nat. Acad. Sci. U.S.A., 1991; 88:3084-3087(Mombaerts et al, Proc. Natl. Acad. Sci. U.S.A.,1991, vol. 88, 3084-3087)).
Antisense, small interfering RNA, DNase, and ribozyme molecules that inhibit expression of a target gene may also be used according to the present invention to reduce the level of target gene activity. For example, antisense RNA molecules that inhibit expression of major histocompatibility gene complexes (HLA) have been shown to be the most versatile for immune responses. In addition, triple helix molecules can be used to reduce the level of target gene activity.
These and other techniques are described in detail in the following documents: L.G. Davis et al, (eds), basic methods In Molecular Biology,2nd ed., Appleton & Lange, Norwalk, Conn. (1994) (L.G. Davis et al, eds., Molecular Biology based methods, 2nd edition, published by Appton and Lange, Norwalk, Conn.), which is incorporated herein by reference.
IL-1 is a potent stimulator of cartilage resorption and the production of inflammatory mediators by chondrocytes (Campbell et al, J.Immun., 1991; 147(4):1238-1246(Campbell et al, J.Immun.,1991, Vol.147, No. 4, p.1238-1246)). Using any of the above techniques, the expression of IL-1 in the cells of the invention can be knocked-out or knocked-down to reduce the risk of resorption by implanted cartilage or production of inflammatory mediators by the cells of the invention. Also, the expression of MHC class II molecules can be knocked out or knocked down in order to reduce the risk of rejection of the implanted tissue.
Once the cells of the invention are genetically engineered, the cells can be implanted directly into a patient for the treatment of a disease or disorder associated with IVD degeneration, for example by producing a product, such as an anti-inflammatory gene product, that has a therapeutic effect on one or more symptoms of the disease or disorder. Alternatively, genetically engineered cells can be used to generate new tissue in vitro, which is then implanted into a subject as described herein.
In some aspects, a pharmaceutical composition comprising umbilical cord tissue-derived cells as described herein and a pharmaceutically acceptable carrier is provided. The pharmaceutical compositions provided herein can induce umbilical cord tissue-derived cells to differentiate along an IVD cellular pathway or lineage, for example, to exhibit a nucleus pulposus cell phenotype and/or an annulus fibrosus cell phenotype. In some embodiments, the pharmaceutical compositions provided herein modulate cellular processes of endogenous IVD cells and/or cells of the surrounding tissue, including but not limited to cell division, differentiation, and gene expression. In some embodiments, the pharmaceutical compositions provided herein promote repair and regeneration of degenerative IVD.
According to a feature of the invention there is also a kit for carrying out the method of the invention. In one aspect, a kit for treating a patient having a disease or injury of at least one IVD is provided. The kit comprises a pharmaceutically acceptable carrier, an amount of cells obtained from human umbilical cord tissue effective to treat the disease or disorder, such as those described and exemplified herein, and instructions for using the kit in a method of treating a patient having a disease or disorder associated with IVD degeneration. The kit may further comprise at least one reagent for culturing cells and instructions for use. The kit may further comprise a population of at least one other cell type and/or at least one agent.
In some aspects, the kit comprises a pharmaceutically acceptable carrier, a lysate, an extracellular matrix, or conditioned medium prepared from cells obtained from human umbilical cord tissue, the cells having the properties described and exemplified herein. The kit may be used to promote repair and/or regeneration of damaged or diseased IVD.
The following examples are provided to describe the invention in more detail. These examples are intended to illustrate, but not to limit, the invention. Also as used in the following examples and elsewhere in this specification, umbilical cord tissue-derived cells useful in the methods of the invention can be isolated and characterized according to the disclosure of U.S. patent nos. 7,510,873 and 7,524,489, which are incorporated by reference in their entirety for their description, isolation and characterization in relation to umbilical cord tissue-derived cells.
Example 1
Isolation of umbilical cord tissue-derived cells
The umbilical cord was obtained from the national institute for disease research interchange (NDRI, philiadelphia, PA) in Philadelphia, PA. The tissue is obtained after normal delivery. The cell separation protocol was performed aseptically in a laminar flow hood. To remove blood and debris, the umbilical cord was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, CA) in the presence of antifungal agents and antibiotics (100 units/ml penicillin, 100 micrograms/ml streptomycin, 0.25 micrograms/ml amphotericin B). The tissue was then incubated in the presence of 50 ml of medium (DMEM-low glucose or DMEM-high glucose; Invitrogen) for 150cm2Mechanical disruption was performed in tissue culture plates until the tissue was cut into a fine slurry. The minced tissue was transferred to 50 ml conical tubes (approximately 5 grams of tissue per tube). The tissue was then digested in DMEM-low glucose medium or DMEM-high glucose medium, each containing an antifungal agent and an antibiotic as described above. In some experiments, an enzyme mixture of collagenase and dispase (DMEM: "C: D" in low glucose medium; collagenase (Sigma, St Louis, Mo.), 500 units/ml; and dispase (Invitrogen, 50 units/ml) was used. In other experiments, a mixture of collagenase, dispase and hyaluronidase ("C: D: H") (DMEM: -collagenase in low glucose, 500 units/ml; dispase, 50 units/ml; and hyaluronidase (Sigma), 5 units/ml) was used. Conical tubes containing tissue, media and digestive enzymes were incubated for 2 hours at 37 ℃ in an orbital shaker at 225rpm (Environ, Brooklyn, NY, inc.) of bruecklin, NY.
After digestion, the tissue was centrifuged at 150 Xg for 5 minutes and the supernatant aspirated. The pellet was resuspended in 20 ml of growth medium (DMEM: Low dextrose (Invitrogen), 15% (by volume) fetal bovine serum (FBS; defined bovine serum; batch number AND 18475; Hyclone, Logan, UT, Utah), 0.001% (by volume) 2-mercaptoethanol (Sigma), 1ml per 100 ml of the antibiotic/antifungal agent described above. the cell suspension was filtered through a 70 micron nylon cell screen (BD Biosciences), an additional 5ml of the rinse containing growth medium was passed through the screen.
The filtrate was resuspended in growth medium (total volume 50 ml) and centrifuged at 150 Xg for 5 minutes. The supernatant was aspirated and the cells were resuspended in 50 ml of fresh growth medium. This process was repeated two more times.
After the last centrifugation, the supernatant was aspirated and the cell pellet was resuspended in 5ml of fresh growth medium. Viable cell numbers were determined using trypan blue staining. The cells are then cultured under standard conditions.
Isolating cells from umbilical cord at 5,000 cells/cm2Inoculating to gelatin-coated T-75cm2In a culture flask (Corning inc., Corning, NY, n.y.) containing the antibiotic/antifungal agent as described above. After 2 days (cells were incubated for 2-4 days in each experiment), spent medium was aspirated from the flask. Cells were washed three times with PBS to remove debris and blood-borne cells. The cells were then supplemented with growth medium and allowed to grow to confluence (about 10 days from passage 0 to passage 1). In subsequent passages (from passage 1 to passage 2, and so on), cells reached near confluence within 4-5 days (75-85% confluence). For these subsequent passages, cells were plated at 5000 cells/cm2And (4) inoculating. Cells were cultured in a humidified incubator at 37 ℃ in 5% carbon dioxide and atmospheric oxygen.
Example 2
By streamingCytological evaluation of cell surface markers of human postpartum origin
Umbilical cord tissue is characterized using flow cytometry to provide a map for identifying cells obtained therefrom.
The cells were cultured in growth medium containing penicillin/streptomycin (Gibco Carlsbad, CA). Cells were cultured in plasma treated T75, T150, and T225 tissue culture flasks (corning, new york) until confluent. The growth surface of the flask was coated with gelatin by incubating 2% (w/v) gelatin (sigma of st louis, missouri) for 20 minutes at room temperature.
Adherent cells in flasks were washed in PBS and detached with trypsin/EDTA. Cells were harvested, centrifuged and concentrated at 1X 10 per ml7Cell concentrations were resuspended in 3% (volume by volume) FBS in PBS. Antibodies to the cell surface markers of interest (see below) were added to 100 microliters of cell suspension according to the manufacturer's instructions and the mixture was incubated at 4 ℃ for 30 minutes in the dark. After incubation, cells were washed with PBS and centrifuged to remove unbound antibody. Cells were resuspended in 500 microliters PBS and analyzed by flow cytometry. Flow cytometry analysis was performed using a FACScalibur instrument (BD Co., San Jose, Becton Dickinson, Calif.).
The following antibodies using cell surface markers:
cells were analyzed at passages 8, 15 and 20 and umbilical cord tissue-derived cells from different donors were compared to each other. In addition, cells cultured on gelatin-coated flasks were compared to cells cultured on uncoated flasks.
Umbilical cord tissue-derived cells show positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr- α, and HLA-A, B, C, as indicated by an increase in fluorescence value relative to an IgG control these cells are negative for detectable expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, as indicated by a fluorescence value comparable to an IgG control.
All cells from passages 8, 15 and 20 expressed CD10, CD13, CD44, CD73, CD90, PDGFr- α and HLA-A, B, C as indicated by fluorescence enhancement relative to IgG controls these cells were negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ as indicated by fluorescence values consistent with IgG controls.
Isolates from individual donors all showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr- α and HLA-A, B, C as reflected by an increase in fluorescence values relative to IgG controls these cells were negative for expression of CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ, with fluorescence values consistent with IgG controls.
Cells expanded on gelatin and uncoated flasks were positive for expression of CD10, CD13, CD44, CD73, CD90, PDGFr- α, and HLA-A, B, C, with increased fluorescence values relative to IgG controls these cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, with fluorescence values consistent with IgG controls.
Thus, umbilical cord tissue-derived cells were positive for CD10, CD13, CD44, CD73, CD90, PDGFr- α, HLA-A, B, C, and negative for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ.
Example 3
Immunohistochemical characterization of cell phenotype
Human umbilical cord Tissue was harvested and soaked overnight at 4 ℃ in 4% (weight to volume) paraformaldehyde immunohistochemical tests were performed using antibodies targeting vimentin (1: 500; sigmA of st louis, missouri), desmin (1:150, prepared by immunizing rabbits; sigmA; or 1:300, prepared by immunizing mice; Chemicon, temkurA, CA), α -smooth muscle actin (SMA; 400; sigmA), cytokeratin 18(CK 18; 1: 400; sigmA), von willebrA factor (vWF; saint: 200; sigmA), and CD34 (human CD34 III; 1: 100; DAKOCytomation (dakocyt cyto, carpiniA, tocatalA, tokyA), additionally tested for in vivo labeling of saxas-36-mo (GRO-1, ct), anti-human biological staining with A microtome (beck microtome), microtome, refrA, refecton, refrA, 100, etc.), and mounted on A microtome (beck-bio-microtome), microtome, refrA, gaku, microscopical technologies (beck, refrA, gaku), microscopical).
Immunohistochemical tests were performed in analogy to previous studies (Messina et al, Exper. neurol., 2003; 184:816-29(Messina et al, J. Experimental neurology, 2003, vol. 184, p. 816-829)). Briefly, tissue sections were washed with Phosphate Buffered Saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (by volume) goat serum (Chemicon, Temankura, Calif.) and 0.3% (by volume) Triton (Triton X-100; Sigma) for 1 hour to access intracellular antigens. Where the epitope of interest is located on the cell surface (CD34, ox-LDL R1), Triton is omitted in all steps of the procedure to prevent epitope loss. In addition, in the case of primary antibodies prepared by immunizing goats (GCP-2, ox-LDL R1, NOGO-A), 3% (by volume) donkey serum was used instead of goat serum throughout the protocol. Primary antibody was diluted in blocking solution, applied to the sections and kept at room temperature for 4 hours. The primary antibody solution was removed and the cultures were washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing blocking agent and goat anti-mouse IgG-Texas Red (1: 250; Molecular Probes, Eugene, OR) and/OR goat anti-rabbit IgG-Alexa 488(1: 250; Molecular Probes) OR donkey anti-goat IgG-FITC (1: 150; St. Cruz Biotech). The culture was washed, and 10. mu.M DAPI (molecular probes) was applied for 10 minutes to develop the cell nucleus.
Fluorescence was observed using an appropriate fluorescence filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, NY) of Melville, NY. Positive staining is expressed as a fluorescence signal that exceeds control staining. Using a colour digital camera and
Vimentin, desmin, SMA, CK18, vWF, and CD34 markers were expressed in subpopulations of cells present within the umbilical cord. In particular, vWF and CD34 expression is restricted to the vessels contained within the umbilical cord. CD34+Cells are located innermost (luminal side). vimentin expression is present in all the matrices and vessels of the umbilical cord SMA is confined to the matrix and outer walls of arteries and veins, but no CK18 and desmin are found in the vessels themselves, desmin is confined to the middle and outer layers.
Example 4
Oligonucleotide array analysis
Using Affymetrix
Human umbilical cords were obtained from the national research exchange for disease (NDRI) of philadelphia, pa after normal term delivery with patient consent. After the tissue is received, the cells are isolated as described above. Cells were cultured in growth medium (using DMEM-LG) on gelatin-coated tissue culture plastic bottles. The culture was incubated at 37 ℃ and 5% CO2And (4) incubating.
Human epidermal fibroblasts were purchased from Cambrex Incorporated (Walkersville, Md.; batch No. 9F0844) and ATCC CRL-1501(CCD39 SK). Both cell lines were cultured in DMEM/F12 medium (Invitrogen, Calsbarda, Calif.) containing 10% (by volume) fetal bovine serum (Hyclone) and penicillin/streptomycin (Invitrogen). Cells were grown on standard tissue-treated plastic.
Human mesenchymal stem cells (hmscs) were purchased from conberts (woxwell, maryland; lots 2F1655, 2F1656, and 2F1657) and cultured in MSCGM medium (conberts) according to the manufacturer's instructions. Cells were incubated at 37 ℃ and 5% CO2Lower growth was on standard tissue cultured plastic.
Human iliac crest bone marrow was received from NDRI with patient consent. Bone marrow was processed according to the method described by Ho et al (WO 2003/025149). Bone marrow was mixed with lysis buffer (155mM NH) at a ratio of 1 part bone marrow to 20 parts lysis buffer4Cl、10mMKHCO3And 0.1mM EDTA, pH 7.2). The cell suspension was vortexed, incubated at ambient temperature for 2 minutes, and centrifuged at 500 × g for 10 minutes. The supernatant was discarded, and the cells were harvestedThe pellet was resuspended in minimal essential medium α (Invitrogen) supplemented with 10% (by volume) fetal bovine serum and 4mM glutamine, the cells were recentrifuged and the cell pellet resuspended in fresh medium, viable monocytes were calculated using Trypan blue stain exclusion (Sigma of St. Louis, Mo.) the monocytes were resuspended at 5X 104 cells/cm2Inoculated in a tissue culture plastic bottle. In the standard atmosphere O2Or 5% O2At 37 ℃ and 5% CO2Cells were incubated. Cells were cultured for 5 days without changing the medium. After 5 days of culture, the medium and non-adherent cells were removed. Adherent cells are maintained in culture.
Actively growing cell cultures were removed from the flask into cold PBS using a cell scraper. Cells were centrifuged at 300 Xg for 5 minutes. The supernatant was removed and the cells were resuspended in fresh PBS and centrifuged again. The supernatant was removed and the cell pellet was immediately frozen at-80 ℃. Cellular mRNA was extracted, transcribed into cDNA, transcribed into cRNA and labeled with biotin. Biotin-labeled cRNA was hybridized to HG-U133A GeneChip oligonucleotide arrays (Affymetrix, Santa Clara CA). Hybridization and data collection were performed according to the manufacturer's instructions. Analysis was performed using "microarray significance analysis" (SAM) version 1.21 computer software (Stanford University; Tusher et al, Proc. Natl. Acad. Sci. USA, 2002; 98:5116-21 (Stanford University; Tusher et al, Proc. Natl. Acad. Sci. 2002; Tusher et al, Proc. Natl. Acad. Sci. 2002; Vol. USA., 2002; Vol. 98, p. 5116-.
Fourteen different cell populations were analyzed. The cells and passage information, culture substrate and culture medium are listed in table 1.
The data were evaluated by analyzing 290 genes differentially expressed in cells by principal component analysis. The analysis allows relative comparisons of inter-population similarity. Table 2 shows the calculated euclidean distances for the comparison cell pairs. The euclidean distance is based on cell comparisons made with 290 genes expressed differentially between different cell types. The euclidean distance is inversely proportional to the similarity between the expression of 290 genes (i.e., the greater the distance, the less similarity exists).
Tables 3 and 4 below show the increased gene expression levels in umbilical cord tissue-derived cells (Table 3), and the decreased gene expression levels in umbilical cord tissue-derived cells (Table 4). One column entitled "probe set ID" refers to the manufacturer's identifier of a set of several oligonucleotide probes located at specific positions on the chip, which probes hybridize to a designated gene ("Gene name" column) comprising a sequence that can be found in the NCBI database under the designation accession number ("NCBI accession number" column).
Table 3: tables showing specific increases in umbilical cord tissue-derived cells compared to other cell lines assayed Quantitative Gene。
Table 4: exhibit reduced expression in cells derived from umbilical cord tissue as compared to other cell lines assayed Gene (a) of (a)。
Tables 5,6 and 7 show genes whose expression levels were increased in human fibroblasts (table 5), ICBM cells (table 6) and MSCs (table 7).
The foregoing assays included cells derived from three different umbilical cords and two different epidermal fibroblast cell lines, three mesenchymal stem cell lines, and three iliac crest bone marrow cell lines. The mRNA expressed by these cells was analyzed using an oligonucleotide array containing probes for 22,000 genes. The results indicated that 290 genes were differentially expressed in these five different cell types. These genes include seven genes whose expression amount is specifically increased in umbilical cord tissue-derived cells. Fifty-four genes were found to have a particular reduced expression level in umbilical cord tissue-derived cells compared to other cell types. Expression of the selected gene was confirmed by PCR. These results demonstrate, for example, that umbilical cord tissue-derived cells have a unique gene expression profile compared to bone marrow-derived cells and fibroblasts.
Example 5
Cell markers in umbilical cord tissue-derived cells
As demonstrated above, the "signature" genes that identify postpartum-derived cells: oxidized LDL receptor 1, interleukin-8, renin, plasma membrane protein, chemokine receptor ligand 3(CXC ligand 3), and granulocyte chemotactic protein 2 (GCP-2). These "signature" genes are expressed at relatively high levels in cells of postpartum origin.
The procedure described in this example was performed to validate microarray data and look for agreement/disagreement between gene and protein expression, as well as to establish a series of reliable assays for detecting unique identifiers of umbilical cord tissue-derived cells.
Umbilical cord tissue-derived cells (four isolates) and normal human epidermal fibroblasts (NHDF; neonatal and adult) were grown in penicillin/streptomycin-containing growth medium in gelatin-coated T75 flasks. Mesenchymal stem cells (MSCGM; Combs, Wolvjinville, Maryland) were grown in a mesenchymal stem cell growth medium set.
For the IL-8 protocol, cells were thawed from liquid nitrogen and at 5,000 cells/cm2Inoculated into a gelatin-coated flask, cultured in a growth medium for 48 hours, and then cultured in 10 ml of a serum-starved medium (DMEM-low glucose (Gibco, Calif.), penicillin/streptomycin (Gibco, Calif.) and 0.1% (w/v) bovine serum albumin (BSA; Sigma, St. Louis, Mo.) for 8 hours. After this treatment, RNA was extracted and the supernatant was centrifuged at 150 × g for 5 minutes to remove cell debris. The supernatant was then frozen at-80 ℃ for ELISA analysis.
Postpartum cells from umbilical cord and human fibroblasts from human neonatal foreskin were cultured in growth medium in gelatin-coated T75 culture flasks. Cells were frozen for 11 passages in liquid nitrogen. Cells were thawed and transferred to 15 ml centrifuge tubes. After centrifugation at 150 Xg for 5 minutes, the supernatant was discarded. Cells were resuspended in 4 ml of medium and counted. Cells were plated at 375,000 cells/flask in 75cm with 15 ml growth medium2The culture was carried out in a flask for 24 hours. The medium was changed to serum-starved medium and cultured for 8 hours. At the end of the incubation, serum starved medium was collected, centrifuged at 14,000 × g for 5 minutes (and stored at-20 ℃).
To estimate the number of cells in each flask, 2 ml of trypsin/EDTA (Gibco, Calif.) was added to each flask. After the cells were isolated from the flask, trypsin activity was neutralized with 8 ml of growth medium. Cells were transferred to 15 ml centrifuge tubes and centrifuged at 150 Xg for 5 minutes. The supernatant was removed and 1ml of growth medium was added to each tube to resuspend the cells. Cell numbers were estimated using a hemocytometer.
The amount of IL-8 secreted by the cells into serum-starved medium was analyzed using an ELISA assay (R & D Systems, Minneapolis, MN). All assays were tested according to the instructions provided by the manufacturer.
RNA was extracted from the confluent umbilical cord tissue-derived cells and fibroblasts, or the expression amount of IL-8 by the cells treated as described above was measured. According to manufacturer's instructions (
RNA was also extracted from human umbilical cord tissue. The tissue (30 mg) was suspended in 700. mu.l of buffer RLT containing 2-mercaptoethanol. Samples were homogenized mechanically and RNA extracted according to the manufacturer's instructions. RNA was extracted with 50. mu.l of DEPC-treated water and stored at-80 ℃. RNA was reverse transcribed using random hexamer primers and TaqMan reverse transcription reagents (Applied Biosystems, Foster City, Calif.) at 25 ℃ for 10 minutes, 37 ℃ for 60 minutes, and 95 ℃ for 10 minutes. The samples were stored at-20 ℃.
Real-time and routine PCR was used to further investigate the recognition of cDNA microarrays as uniquely regulated genes (signature genes-including oxidized LDL receptor, interleukin-8, renin and plasma membrane proteins) in postpartum cells.
The Assys-on-Demand was made according to the manufacturer's instructionsTMThe gene expression product: oxidized LDL receptor (Hs 00234028); renin (Hs 00166915); serosal protein (Hs 00382515); CXC ligand 3(Hs 00171061); GCP-2(Hs 00605742); IL-8(Hs 001)74103) (ii) a And GAPDH (applied biosystems, Foster City, Calif.) was mixed with cDNA and TaqMan Universal PCR amplification premix reagents (TaqMan Universal PCR master mix), and PCR was performed on the cDNA samples using the 7000 sequence detection System (applied biosystems, Foster City, Calif.) with ABI Prism 7000SDS software (applied biosystems, Foster City, Calif.). The thermal cycling conditions were initially 50 ℃ for 2 minutes and 95 ℃ for 10 minutes, followed by 40 cycles of 95 ℃ for 15 seconds and 60 ℃ for 1 minute. The PCR data were analyzed according to the manufacturer's instructions (user manual #2 for ABI Prism 7700 sequence detection System from applied biosystems).
Conventional PCR was performed using ABI PRISM 7700 (Perkin Elmer Applied Biosystems, Boston, Mass., USA)) to verify the results obtained by real-time PCR. PCR was performed using 2. mu.l of cDNA solution, 1 × AmpliTaq Gold Universal mix PCR reaction buffer (applied biosystems, Foster City, Calif.) and initial denaturation at 94 ℃ for 5 minutes. Amplification was optimized for each primer pair. For IL-8, CXC ligand 3 and plasma membrane protein (94 ℃ 15 seconds, 55 ℃ 15 seconds and 72 ℃ 30 seconds for 30 cycles); for renin (38 cycles at 94 ℃ for 15 seconds, 53 ℃ for 15 seconds, and 72 ℃ for 30 seconds); oxidized LDL receptor and GAPDH (94 ℃ for 15 seconds, 55 ℃ for 15 seconds and 72 ℃ for 30 seconds for 33 cycles). The primers used for amplification are listed in table 8. The primer concentration in the final PCR reaction was 1 micromolar except for GAPDH, which was 0.5 micromolar. The GAPDH primers were identical to the real-time PCR except that the manufacturer's TaqMan probe was not added to the final PCR reaction. The samples were run on a 2% (w/v) agarose gel and stained with ethidium bromide (sigma of st louis, missouri). Images were acquired with a 667Universal Twinpack film (VWRITE International, NJ) and a Focus Baoli (Polaroid) camera (VWR International, NJ).
Table 8: primers used
Cells were fixed for 10 minutes at room temperature using cold 4% (w/v) paraformaldehyde (Sigma-Aldrich, st. louis, MO), immunocytochemistry was performed using one isolate of passage 0 (P0) (used directly after isolation) and two isolates of passage 11 (P11) and fibroblasts (P11), using antibodies targeting epitopes vimentin (1:500, Sigma of st. louis, MO), desmin (1: 150; Sigma-prepared by immunizing rabbits, or 1: 300; saint chemie of tmania cheilon, ca-prepared by immunizing mice), smooth muscle actin (SMA; 1: 400; Sigma), cytokeratin 18(CK 18; 1: 400; Sigma), vascular factor (vWF; 1: 200; Sigma) and CD 2 (CD 2; CD 2: cth; gck III; cykl 1: 100: ga), anti-human biol technologies of anti-human biol 1: 11 (g) and anti human biol-a technologies (gry) and anti human biol 1:100 g 1: 11 (g-kohlaji) technologies.
Cultures were washed with Phosphate Buffered Saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (by volume) goat serum (Chemicon, Temankura, Calif.) and 0.3% (by volume) Triton (Triton X-100; Sigma, St.Louis, Mo.) for 30 minutes to gain access to intracellular antigens. Where the epitope of interest is located on the cell surface (CD34, ox-LDL R1), Triton X-100 is omitted at all steps of the procedure to prevent epitope loss. In addition, in the case of primary antibodies prepared by immunizing goats (GCP-2, ox-LDL R1, NOGO-A), 3% (by volume) donkey serum was used instead of goat serum throughout the process. After dilution in blocking solution, primary antibody was added to the culture and kept at room temperature for 1 hour. The primary antibody solution was removed and the cultures were washed with PBS before applying a secondary antibody solution (1 hour at room temperature) containing the blocking agent and either goat anti-mouse IgG-Texas Red (1: 250; molecular probes, Yougu, Oregon) and/or goat anti-rabbit IgG-Alexa 488(1: 250; molecular probes) or donkey anti-goat IgG-FITC (1:150, Santa Cruz Biotech). The culture was then washed and 10. mu.M DAPI (molecular probes) was applied for 10 minutes to develop the nuclei.
After immunostaining, fluorescence was observed using an appropriate olympus inverted epifluorescence microscope (olympus, melville, ny). In all cases, a positive stain indicates a fluorescence signal that exceeds the control stain, with all procedures described above being followed except for the application of the primary antibody solution. Using a colour digital camera andthe software (Media Cybernetics, Inc. of Calsbad, Calif.) collects representative images. For the triple stained samples, each image was taken with only one emission filter at a time. The layered clips were made using Adobe Photoshop software (Adobe, San Jose, Calif.).
Adherent cells in flasks were washed in Phosphate Buffered Saline (PBS) (Gibco, Calsbad, Calif.) and detached with trypsin/EDTA (Gibco, Calsbad, Calif.). Cells were harvested, centrifuged and concentrated at 1X 10 per ml7Cell concentrations were resuspended in 3% (volume by volume) FBS in PBS. One hundred microliter aliquots were delivered into conical tubes. Cells stained for intracellular antigens were permeabilized using a membrane-breaking/washing buffer (BDPharmingen, san diego, ca). Antibodies were added to aliquots according to the manufacturer's instructions and cells were incubated for 30 minutes in the dark at 4 ℃. After incubation, cells were washed with PBS and centrifuged to remove excess antibody. Cells requiring secondary antibody were resuspended in 100 microliters of 3% FBS. Secondary antibodies were added according to the manufacturer's instructions and cells were incubated for 30 minutes at 4 ℃ in the dark. After incubation, cells were washed with PBS and centrifuged to remove excess secondary antibody. The washed cells were resuspended in 0.5ml PBS andanalysis was performed by flow cytometry. The following antibodies were used: oxidized LDL receptor 1 (sc-5813; santa cruz biotechnology), GROa (555042; BD Pharmingen (BDPharmingen, Bedford, MA), mouse IgG1 κ (P-4685 and M-5284; sigma), donkey anti-goat IgG (sc-3743; santa cruz biotechnology). Flow cytometry analysis was performed using FACScalibur (BD Co., san Jose, Calif.).
Data from real-time PCR were analyzed by the Δ Δ CT method and expressed on a logarithmic scale. The levels of plasma membrane protein and oxidized LDL receptor expression are higher in umbilical cord tissue-derived cells than in other cells. No significant difference in the expression levels of CXC ligand 3 and GCP-2 was found between postpartum-derived cells and controls. The results of real-time PCR were verified by conventional PCR. Sequencing of the PCR products further validated these observations. No significant differences in the expression levels of CXC ligand 3 were found between postpartum-derived cells and controls using the conventional PCR CXC ligand 3 primers listed above.
The production of the cytokine IL-8 in postpartum cells is elevated in growth medium cultured and serum starved postpartum-derived cells. All real-time PCR data was verified using conventional PCR and by sequencing the PCR products.
The highest amounts were detected in the media of some isolates derived from umbilical cord cells and placental cells when the presence of IL-8 was detected in the supernatant of cells grown in serum-free media (Table 9). No IL-8 was detected in the medium derived from human epidermal fibroblasts.
Following isolation (passage 0), cells were fixed with 4% paraformaldehyde and exposed to antibodies to six proteins, von willebrand factor, CD34, cytokeratin 18, desmin, α -smooth muscle actin and vimentin.
The agreement between the gene expression levels of the four genes, oxidized LDL receptor 1, renin, serosal protein and IL-8, has been determined by microarray and PCR (both real-time and routine). the expression of these genes is differentially regulated at the mRNA level of PPDC, and IL-8 is also differentially regulated at the protein level.
Example 6
In vitro immunological evaluation of postpartum derived cells
The immunological Profile of Postpartum Derived Cells (PPDCs) was evaluated in vitro to predict whether these cells will elicit any immune response upon transplantation in vivo. PPDC was analyzed by flow cytometry for the presence of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed by Antigen Presenting Cells (APCs) and are the primary CD4+Required for direct T cell stimulation (Abbas)&Lichtman, Cellular and Molecular Immunology,5th Ed. (Saunders, philiadelphia, 2003; p.171) (Abbas and Lichtman, cell and Molecular Immunology,5th edition, (sanders, Philadelphia, 2003; page 171)). The cell lines were also analyzed by flow cytometry for expression of HLA-G (Abbas and Lichtman, 2003, supra), CD178 (Coumans, et al, Journal of immunological methods, 1999; 224:185-196(Coumans et al, J. Immunol. methods,1999, vol. 224, pp. 185-196)) and PD-L2(Abbas and Lichtman, 2003, supra; Brown, et al, The Journal of immunology, 2003; 170:1257-1266(Brown et al, J. Immunol.,2003, vol. 170, pp. 1257-1266)). Expression of these proteins by cells present in placental tissue is believed to mediate the immune-privileged state of placental tissue in utero. To predict the extent to which placental and umbilical cord tissue-derived cell lines elicit an immune response in vivo, the cell lines were tested in a one-way Mixed Lymphocyte Reaction (MLR).
The cells were cultured to confluence in penicillin/streptomycin-containing growth medium in T75 flasks (corning, n.y.) coated with 2% gelatin (sigma, st. louis, missouri).
Cells were washed in Phosphate Buffered Saline (PBS) (Gibco, carlsbad, california) and detached with trypsin/EDTA (Gibco, carlsbad, missouri). Cells were harvested, centrifuged and concentrated at 1X 10 per ml7Cell concentrations were resuspended in 3% (volume by volume) FBS in PBS. Antibodies (table 10) were added to 100 microliters of cell suspension according to the manufacturer's instructions and incubated at 4 ℃ for 30 minutes in the dark. After incubation, cells were washed with PBS and centrifuged to remove unbound antibody. Cells were resuspended in 500 microliters PBS and analyzed by flow cytometry using a FACSCalibur instrument (BD, san jose, california).
Frozen storage vials of 10 passages of umbilical cord tissue-derived cells labeled cell line a were shipped on dry ice to CTBR company (CTBR (Senneville, Quebec)) in turnera, Quebec, to perform mixed lymphocyte reactions using CTBR SOP No. cac-031. A plurality of Peripheral Blood Mononuclear Cells (PBMCs) are collected from a plurality of male and female volunteer donors. Stimulation factor (donor) allergic PBMC, autologous PBMC and postpartum cell lines were treated with mitomycin C. Autologous and mitomycin C-treated stimulatory cells were added to responsive (recipient) PBMC and cultured for 4 days. After incubation, the [ 2 ], [3H]Thymidine was added to each sample and cultured for 18 hours. After harvesting the cells, the radiolabeled DNA is extracted and measured using a scintillation counter3H]-thymidine incorporation.
The stimulation index of the heterologous donor (SIAD) was calculated as the average proliferation rate of the recipient plus mitomycin C treated heterologous donor divided by the baseline proliferation rate of the recipient. The stimulation index of PPDC was calculated as the mean proliferation rate of the recipient mitomycin C-treated postpartum cell line divided by the baseline proliferation rate of the recipient.
Six volunteer donors were screened to identify a single heterogeneous donor that would exhibit a robust proliferative response in a mixed lymphocyte reaction with the other five donors. This donor was selected as a heterologous positive control donor. The remaining five donors were selected as recipients. The heterologous positive control donor and placental cell lines were treated with mitomycin and cultured in mixed lymphocyte reactions to react with five separate heterologous recipients. Reactions were performed in triplicate using two cell culture plates with three recipients per plate (table 11). The mean stimulation index ranged from 6.5 (plate 1) to 9 (plate 2) and the allogeneic donor positive control ranged from 42.75 (plate 1) to 70 (plate 2) (table 12).
Table 11: mixed lymphocyte reaction data-cell line A (umbilical cord)
DPM for proliferation assays
Plate ID: panel 1
Plate ID: plate 2
Table 12: umbilical cord tissue-derived cells and xenogeneic in a mixed lymphocyte reaction with five separate allogeneic recipients Mean stimulation index of Source donors。
Mean stimulation index
Receptors
Umbilical cord
Panel 1 (recipients 1-4)
42.75
6.5
Plate 2 (receiver 5)
70
9
Flow cytometry analysis of the resulting histograms of umbilical cord tissue-derived cells demonstrated negative expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as indicated by fluorescence values consistent with IgG controls, indicating the lack of direct stimulation of the umbilical cord cell line with CD4+Cell surface molecules required for T cells. Flow cytometry analysis of the resulting histogram of umbilical cord tissue-derived cells demonstrated positive expression of PD-L2, as indicated by an increase in fluorescence relative to the IgG control, and demonstrated negative expression of CD178 and HLA-G, as indicated by fluorescence consistent with the IgG control.
In the mixed lymphocyte reaction with the umbilical cord tissue-derived cell line, the average stimulation index was in the range of 6.5 to 9, and the average stimulation index of the heterologous positive control was in the range of 42.75 to 70. The umbilical cord tissue-derived cell lines were negative for expression of the stimulatory proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 as determined by flow cytometry. The umbilical cord tissue-derived cell line was negative for the expression of the immunomodulatory proteins HLA-G and CD178, and positive for the expression of PD-L2, as determined by flow cytometry. Heterologous donor PBMC comprise antigen presenting cells expressing HLA-DR, DQ, CD8, CD86 and B7-H2, allowing stimulation of primary CD4+T cells. Lack of primary CD4 on placenta and umbilical cord tissue-derived cells+Direct T cell stimulation of the desired antigen presenting cell surface molecule and the presence of the immunomodulatory protein PD-L2 results in this in comparison to a heterologous controlSome cells showed low stimulation index in MLR.
Example 7
Secretion of trophic factors by umbilical cord tissue-derived cells
Factors selected for detection include (1) those known to have angiogenic activity, such as Hepatocyte Growth Factor (HGF) (Rosen et al, Ciba Found, Symp., 1997; 212:215-26(Rosen et al, Ciba Foundation conference, 1997, Vol 212, p.215), monocyte chemotactic protein 1(MCP-1) (Salcedo et al, Blood, (2000; 96:34-40(Salcedo et al, Blood, 2000, Vol 94, p.34-40)), interleukin-8 (IL-8) (Li et al, J.Immunol., 2003; 170:3369-76(Li 2004 et al, J.Immunol., 2003; J.J.Immunol., 2003; 170, p.3369-76 (Li et al, J.J.J.J.J.J.J.J.J.J.J.J.P.P.P.103, J.P.J.J.P.J.J.J.J.J.J.EP., 2003, 3369, p.p.p.p.p.35), thrombopoietin.7, VEGF-2, VEGF-activating factor (VEGF-2), growth promoting factor (VEGF-activating protein), growth promoting factor), growth factor (VEGF-activating protein), growth factor activating protein (VEGF-activating protein), VEGF-activating protein (VEGF-activating protein), growth factor activating protein (VEGF-activating protein), protein activating protein (VEGF-activating protein), protein activating protein (VEGF-activating protein (VEGF-activating protein), protein activating.
Culturing cells from umbilical cord and human fibroblasts from human neonatal foreskin in gelatin-coated T75 culture flasksGrowth medium containing penicillin/streptomycin. Cells were frozen for 11 passages and stored in liquid nitrogen. After the cells were thawed, growth medium was added to the cells, which were then transferred to a 15 ml centrifuge tube and the cells were centrifuged at 150 × g for 5 minutes. The supernatant was discarded. The cell pellet was resuspended in 4 ml growth medium and the cells were counted. At 375,000 cells/75 cm2The flasks containing 15 ml of growth medium were inoculated with cells and cultured for 24 hours. The medium was replaced with serum-free medium (DMEM-Low glucose (Gibco), 0.1% (w/v) bovine serum albumin (Sigma), penicillin/streptomycin (Gibco)) and cultured for 8 hours. Conditioned serum-free medium was collected at the end of the incubation by centrifugation at 14,000 Xg for 5 minutes and stored at-20 ℃. To estimate the number of cells in each flask, the cells were washed with PBS and detached using 2 ml trypsin/EDTA. Trypsin activity was inhibited by addition of 8 ml growth medium. Cells were centrifuged at 150 Xg for 5 minutes. The supernatant was removed and the cells were resuspended in 1ml growth medium. Cell numbers were estimated using a hemocytometer.
Cells were cultured in 5% carbon dioxide and atmospheric oxygen at 37 deg.C placenta-derived cells (batch No. 101503) were also cultured in 5% oxygen or β -mercaptoethanol (BME). the amount of MCP-1, IL-6, VEGF, SDF-1 α, GCP-2, IL-8, and TGF- β 2 produced by each cell sample was determined by ELISA assays (Andy organisms, Minneapolis, Minn.).
Use of
MCP-1 and IL-6 were secreted by umbilical cord tissue-derived cells and epidermal fibroblasts (Table 13). SDF-1 α was secreted by fibroblasts GCP-2 and IL-8 were secreted by BME or 5% O2GCP-2 is also secreted by human fibroblasts, TGF- β 2 was not detectable by the ELISA assay.
TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC and IL-8 were secreted from umbilical cord tissue-derived cells (tables 14 and 15). Ang2, VEGF, or PDGF-bb were not detected.
Umbilical cord tissue-derived cells secrete a variety of very beneficial trophic factors. Some of these trophic factors, such as the catabolic inhibitor TIMP1, play a key role in preventing matrix metalloproteinases from degrading the extracellular matrix. HGF, bFGF, MCP-1 and IL-8 play important roles in cell survival and cell differentiation functions. Other trophic factors such as BDNF and IL-6 play important roles in nerve regeneration.
Example 8
IFN-alpha of HMG-CoA reductase inhibitor to HLA-DR, DP, DQ on expanded human umbilical cord tissue-derived cells Inhibition of gamma-induced expression
Cultured expanded human umbilical cord tissue-derived cells (022803P4) were seeded into 6-well tissue culture plates and grown to approximately 70% confluence in Dulbecco' S modified Eagles medium (DMEM) -low glucose, 15% Fetal Bovine Serum (FBS), penicillin/streptomycin (P/S), β mercaptoethanol (BME), then cells were treated with media containing 10 μ M of the respective HMG-CoA reductase inhibitor (simvastatin acid (Alexis Biochemicals of Lausen, Switzerland)) formulated as 10mM stock reagent in DMSO or DMSO-0.1% (sigma-CoA of saint louisis, missou) and incubated overnight, the media was removed by aspiration and replaced with media containing 500U/ml of rhIFN- γ (BD lake of franklin, new jersey) and 10 μ M HMG-CoA inhibitor, and cells were harvested on day 3.
The harvested cells were washed once with PBS and resuspended in 100 μ l of 3% FBS in PBS containing 20 μ l of FITC-labeled HLA-DR, DP, DQ (BD bioscience division of franklin lake, new jersey) or FITC-labeled IgG antibody (BD bioscience division of franklin lake, new jersey) and incubated for 1 hour. Cells were washed once with PBS, resuspended in 500 μ Ι PBS, and analyzed on a FACSCalibur flow cytometer (BS Biosciences, Franklin Lakes, NJ).
As shown in table 16, untreated and 0.1% DMSO vehicle control human umbilical cord tissue-derived cells incubated with the inflammatory cytokine IFN- γ showed an increase in HLA-DR, DP, DQ expression as seen by increased fluorescence detected by flow cytometry. Human umbilical cord tissue-derived cells pretreated with HMG-CoA reductase inhibitor and subsequently incubated with IFN- γ showed similar HLA-DR, DP, DQ expression as untreated and vehicle controls.
This data shows that HMG-CoA reductase inhibits inflammatory cytokine-mediated expression of HLA-DR, DP, DQ in human umbilical cord tissue-derived cells.
Example 9
Efficacy of human umbilical cord tissue-derived cells (hUTC) in Rabbit model of intervertebral disc degeneration
A study was conducted to determine whether human umbilical cord tissue-derived cells (hUTC) were effective in a rabbit model of intervertebral disc (IVD) degeneration. Cells were injected at the site of injury and disc size was assessed by X-ray imaging. Histological analysis was performed at necropsy.
To assess the effect of human umbilical cord tissue-derived cells (hUTC) on degeneration of intervertebral discs (IVD), hUTC was injected into the punctured IVD. X-ray images were taken every two weeks and analyzed for changes in disc height compared to the injured vehicle-treated control. Treatment with hUTC resulted in increased disc height and increased recovery rate compared to vehicle control.
Constructing an intervertebral disc degeneration model: female NZW rabbits were selected for 6 months of age without any systematic bias. Animals were labeled and weighed prior to selection and immediately prior to necropsy. Rabbits received glycopyrrolate(s) (subcutaneous injections 0.01 to 0.02mg/kg) prior to sedation to reduce orotracheal secretions and relieve bradycardia associated with anesthesia. Buprenorphine (buprenorphine hydrochloride 0.03mg/kg) was administered as a pre-analgesic prior to surgery. Rabbits were anesthetized by administration of ketamine hydrochloride (25mg/kg) and acepromazine maleate (1mg/kg,10mg/ml) for endotracheal intubation. Pre-operative X-ray images were taken as baseline controls. A5 mg/kg dose of xylazine was administered subcutaneously or intramuscularly after the pre-operative photographs were completed. Animals were maintained with isoflurane inhalant (induced at 2-3% and maintained at 0.5-2%).
The rabbit (weighing 3.5kg) was in a lateral position. Following sterilization and drape, lumbar IVD is exposed via posterolateral retroperitoneal approach by blunt separation of the psoas major muscles. The front surfaces of three consecutive waists IVD (L2/3, L3/4 and L4/5) are exposed. The annulus was punctured ventrally into the nucleus at sections L2/3 and L4/5 using an 18G needle with a blocking device that allowed the needle to pass 5mm deep. Vascular staples and sutures were placed on the psoas major muscle at segment L3/4 as a marker. The formed surgical wounds are repaired in a layered manner. The skin is closed using staples.
After surgery, post-operative X-ray images were taken to verify the level of penetration. 1.5mg of meloxicam was administered orally (one day before surgery and 2-3 days after surgery). The analgesic (buprenorphine hydrochloride 0.01-0.03 mg/kg) may be administered as required up to two times daily for 2-3 days in the case of consultations with veterinary personnel. After recovery from anesthesia, the rabbits were returned to their cages and allowed to move freely. Rabbit coats are used on rabbits to prevent them from contacting and disturbing/tearing the surgical incision and removing the staples.
And (3) treatment evaluation: around the time after the initial surgery (circular puncture), a similar surgery was performed from the opposite side to avoid bleeding from the scar formed by the first surgery. When surgically degenerated discs were confirmed by X-ray and visual observation, PBS, 1,000,000 or 100,000 cells were injected intradiscally into the nucleus pulposus region using a microinjector using a 28G needle at L2/3 and L4/5 sections of each rabbit. L3/4 was left as a no puncture, untreated control. After surgical wound repair, the rabbits were returned to their cages and monitored closely. Antibiotics and analgesics were administered for three days as described previously. Each rabbit received 1.5mg meloxicam orally (one day before surgery and 2-3 days after surgery). Behavioral, appetite and weight changes were closely monitored, and veterinarians and researchers monitored post-operative stress.
All injection materials were prepared under sterile conditions. A research grade hUTC (batch No. Q030306) that has been evaluated for sterility, mycoplasma, karyotype, and pathogens was used in this study. Cryopreserved cells were quickly thawed and diluted in PBS. The cells were centrifuged and the supernatant removed. Cells were resuspended in PBS and counted to obtain final cell concentrations of 100,000 or 10,000 cells per microliter. Viability was assessed using trypan blue. 10 microliters of cells were loaded into a pre-sterilized microsyringe and injected into the IVD as described above.
At 2, 4, 6, 8, 10, 12, 14 and 16 weeks post-puncture and at 16 weeks post-puncture at time of death, X-ray images were taken to measure IVD height after ketamine hydrochloride (25mg/kg) and acepromazine maleate (1mg/kg) administration.
At 16 weeks after the initial circular puncture (corresponding to 12 weeks after injection (of cells)), eight rabbits in each group were anesthetized with ketamine hydrochloride (25mg/kg) and acepromazine maleate (1mg/kg) and euthanized with an overdose of pentobarbital (90mg/kg, euthanized B solution: Henry Schein inc, Melville, NY).
X-ray films obtained before the puncture, at each time point after the puncture and at the time of euthanasia were digitized, and the vertebral body height and the intervertebral disc height were measured. IVD height is expressed as DHI according to published methods (Chujo et al, Spine, vol. 31, p. 2909-2917; Chujo et al, Spine, vol. 2909-2917; Masuda et al, Spine, vol. 2006, 31:742-54(Masuda et al, Spine, vol. 2006, vol. 31, p. 742-754)). An orthopedic researcher independently interprets all X-ray images without knowing the treatment group. The digitized X-rays, measurements, including vertebral body height and IVD height, were analyzed using a custom program of MATLAB software (Natick, MA, massachusetts). The data were transferred to Excel software and the IVD height was expressed as an intervertebral disc height index (DHI-IVD height/adjacent IVD body height) according to the method of Lu et al, Spine,22:1828-34(1997) with a slight modification (Lu et al, spinal column, Vol.22, p. 1828-1834, 1997). The average IVD height (DHI) is calculated by averaging measurements taken from the anterior, medial and posterior IVD, divided by the average of the adjacent vertebral body heights. The% DHI is expressed as (postoperative DHI/preoperative DHI). times.100. In addition, the% DHI was normalized using the L3/4 segment as a control segment. Normalized% DHI ═ (experimental section% DHI/L3/4% DHI) × 100. The recovery rate is also calculated as follows: (% DHI (time point) -% DHI (4W [ puncture))/(100-% DHI (4W [ puncture ])).
Significance of differences between the mean values of the X-ray measurement-related data was analyzed by two-way repeated measures ANOVA or one-way ANOVA and Fisher's PLSD as post hoc tests. All data are expressed as mean ± standard deviation. Statistical analysis was performed with a significance level of p <0.05 using the Statview (version 5.0, SPSS, chicago, illinois) package.
The disc height index was evaluated every two weeks as described above. Discs with less than 10% defects were excluded from the study. The data (shown in Table 17) indicate that doses of hUTC at 100,000 cells increased disc height, while doses of 1,000,000 cells decreased disc height (p <0.05, hUTC (1,000,000) versus hUTC (100,000) at 2, 4, 6 and 14 weeks post-implantation; p <0.01, 8 weeks post-implantation) as compared to controls between 2-12 weeks post-implantation (6-16 weeks post-puncture).
The recovery rate was determined as described above. Discs with less than 10% defects were excluded from the study. The data (shown in table 18) show that doses of hUTC with 100,000 cells increase the recovery rate, while doses of 1,000,000 decrease the recovery rate, as compared to controls between 2-12 weeks post-transplantation (6-16 weeks post-puncture). (p <0.05, control versus hUTC (100,000 cells) 2 and 14 weeks after transplantation; p <0.01, 12 weeks after transplantation).
Thus, the effect of hUTC on IVD degeneration was evaluated in a rabbit model of IVD degeneration by injecting hUTC into the spiked IVD at doses of 1,000,000 and 100,000 cells/injection. X-ray images were taken every two weeks and analyzed for changes in disc height compared to untreated puncture controls. The data show that treatment with 100,000 concentration of hUTC resulted in an increase in disc height, while treatment with 1,000,000 concentration resulted in a decrease in disc height, as compared to PBS-treated controls (table 17). Further analysis of the data indicated that the recovery rate (normalized% recovery/total loss of DHI) was higher for discs treated with hUTC (100,000) than for the control (Table 18).
Example 10
Expression of extracellular matrix proteins by human umbilical cord tissue-derived cells in vitro
The cells were tested individually and after stimulation with trophic factors, TGF- β, GDF-5, and PDGF-BB.
Briefly, cells were seeded at 5000 cells per square centimeter in T-flasks and passaged and reseeded every 3-4 days. cells for trophic factor experiments were encapsulated in alginate microspheres and treated with factors in standard growth media.100. mu.g/ml ascorbic acid was supplemented to the culture.the factors tested were PDGF-BB at 10ng/ml, TGF β -1 at 5ng/ml, and GDF-5 at 200 ng/ml.
After 2 weeks of culture, cells were released from alginate, washed, pelleted and frozen. RNA was isolated and reverse transcribed to produce cDNA. Samples were analyzed by real-time PCR for expression of aggrecan, collagen type I and type II.
The results obtained from real-time PCR analysis show expression under five different culture conditions the results are shown in Table 19 and expressed as relative expression to hUTC in the absence of growth factor treatment cultures treated with PDGF-BB and GDF-5 with TGF- β 1 showed comparable expression levels of aggrecan, collagen I and collagen II cells treated with TGF- β 1 showed a certain level of induction of collagen I and collagen II and aggrecan of about 10-20 fold, the highest level of induction observed when treated with GDF-5 cells showed about a 50-fold increase in aggrecan and collagen type I and more than a 300-fold increase in collagen type II expression.
Example 11
Telomerase expression in umbilical cord-derived cells
Telomerase acts to synthesize telomeric repeats that serve to protect chromosomal integrity and extend the replicative life of cells (Liu, K, et al, PNAS, 1999; 96: 5147-. Telomerase consists of two components, the telomerase RNA template (hTER) and the telomerase reverse transcriptase (hTERT). The regulation of telomerase is determined by the transcription of hTER, not hTERT. Real-time Polymerase Chain Reaction (PCR) of hTERT mRNA is therefore an acceptable method for determining telomerase activity of cells.
And (5) separating the cells. Real-time PCR experiments were performed to determine telomerase production by human umbilical cord tissue-derived cells. Human umbilical cord tissue-derived cells were prepared according to the above experiment. Generally, umbilical cord obtained from the national disease research exchange of philadelphia, pennsylvania after normal childbirth is washed to remove blood and cellular debris and mechanically dissociated. The tissue is then incubated with digestive enzymes (including collagenase, dispase, and hyaluronidase) in culture medium at 37 ℃. Human umbilical cord tissue-derived cells were cultured according to the methods described in the examples above. Mesenchymal stem cells and normal epidermal fibroblasts (cc-2509 batch 9F0844) were obtained from Combers, Wolville, Maryland. Pluripotent human testicular embryonic carcinoma (teratoma) cell line nTera-2 Cells (NTERA-2cl. Dl) (see, Plaia et al, Stem Cells, 2006; 24(3):531-546(Plaia et al, Stem Cells,2006, Vol. 24, No. 3, p. 531-546)) were purchased from the American type culture Collection of Manassas, Va.) and cultured according to the method described above.
And (4) separating total RNA. Use of
And (5) carrying out real-time PCR. Using applied biosystems according to manufacturer's instructions (applied biosystems company)(also referred to as
hTERT and 18S RNA was determined for human umbilical cord tissue-derived cells (ATCC accession No. PTA-6067), fibroblasts, and mesenchymal stem cells. As shown in table 20, hTERT and thus telomerase was not detected in human umbilical cord tissue-derived cells.
Human umbilical cord tissue-derived cells (isolate 022803, ATCC accession No. PTA-6067) and nTera-2 cells were assayed and the results indicated that neither batch of human umbilical cord tissue-derived cells expressed telomerase while the teratoma cell line showed high levels of expression (table 21).
Therefore, the following conclusions can be drawn: the human umbilical cord tissue-derived cells of the present invention do not express telomerase.
Example 12
Human umbilical cordInjection of tissue-derived cells into the nucleus pulposus alters the process of intervertebral disc degeneration in vivo
This example investigates the utility of injecting human umbilical cord tissue-derived cells (hUTC) directly into the Nucleus Pulposus (NP) in a surgical in vivo model of IVD degeneration. hUTC was injected with and without a hydrogel carrier. The study led to the study of MRI and biomechanical responses to treatments in this degenerative model.
Method of producing a composite material
As will be described in more detail below, thirty new zealand white rabbits with mature bones (control n-6, puncture n-6, hydrogel carrier n-6, cell + PBS buffer n-6, cell + hydrogel carrier n-6) were used in the previously validated rabbit-ring-dissection model of intervertebral disc degeneration (see Sobajima et al spine.2005; 30(1):15-24(Sobajima et al, spine, 2005, vol.30, No. 1, p.15-24)). Intervertebral discs L2-3, L3-4 and L4-5 were punctured with a 16G needle to induce degeneration, and then treated with human umbilical cord tissue-derived cells with or without a hydrogel carrier. Serial spinal MRIs obtained at 0,3, 6 and 12 weeks using a 3T knee coil were analyzed according to previously validated methods to confirm degeneration. Rabbits were sacrificed at 12 weeks and disc L4-5 was histologically analyzed. The viscoelastic properties of the L3-4 disc were analyzed using a uniaxial load normalized displacement test. The creep curve is mathematically modeled according to a previously validated two-phase exponential model.
Rabbit
Thirty healthy skeletal matured new zealand white rabbits (female, age 1, body weight 5kg) were used in this study. These were divided into a non-puncture control (n-6), a puncture group (n-6) after injection of the vector alone, a puncture group (n-6) after injection of the cells in PBS buffer, and a puncture group (n-6) after injection of the cells in the vector.
Sample preparation
Human umbilical cord tissue was obtained from consented donors who underwent vaginal or caesarean delivery. Human umbilical cord tissue-derived cells (hUTC) were isolated and expanded from umbilical cords of individual donors. Briefly, the cord was manually minced and treated with 0.5 units/ml collagenase (Nor)dmark)), 5.0 units/ml dispase (Roche Diagnostics), and 2 units/ml hyaluronidase (ISTA Pharmaceuticals) until near complete digestion. The cell suspension was passed through a sieve to remove undigested tissue, and the cells were centrifuged and expressed per cm2Growth medium 1 (DMEM-Low glucose (Combbios/SAFC Biosciences)), 15% (volume ratio) defined fetal bovine serum (FBS; Hyclone of Rogen, Utah), 0.001% β mercaptoethanol (Sigma), 50U/ml penicillin, and 50 μ g/ml streptomycin (Longza)). cells were expanded in static T flasks at atmospheric oxygen, 5% carbon dioxide, and 37 ℃ under standard tissue culture conditions until five Population Doublings (PD) were achieved and stored.cells were then expanded in growth medium 2 (DMEM-Low glucose, 15% (volume ratio) defined fetal bovine serum (FBS; Hyclone), 100U/ml penicillin, and 100 μ g/ml streptomycin (Invitrogen)) in static T flasks to approximately 12 PD and then stored.cells were allowed to accumulate in growth medium 2 (DMEM-Low glucose, 15% (volume ratio) defined fetal bovine serum (FBS; Hyclone), 100U/ml penicillin, and 100 μ g/ml streptomycin Ninggen (Invitrogen) flasks
Puncture surgery
In a veterinary operating room, the rabbit's lumbar spine was exposed from a left antero-lateral approach under sterile surgical conditions and general anesthesia. The L4-5 disc was determined by its position relative to the iliac crest and was pierced with a 16 gauge needle to a depth of 5mm so that the tip of the needle was in the center of the disc. The needle enters the disc in a direction parallel to the endplates with the bevel pointing cephalad. Discs L3-4 and L2-3 were then identified by direct visualization and palpation and punctured as above. After surgery, rabbits were housed in large individual cages, leaving their mobility unrestricted. Loop dissection of rabbit IVD by this method induced a slow, reliable, repeatable degenerative cascade, as demonstrated by sequential T2-weighted MRI (see Masuda et al spine.2005; 30(1):5-14(Masuda et al, Spine.2005, Vol. 30, No. 1, p. 5-14)).
Injection surgery
Rabbits in the treatment group were again surgically operated 3 weeks after the initial stab surgery. Exposing the spine from the right antero-lateral approach (contralateral to the previous stab surgery). Hamilton (Hamilton)100 microliter syringe (1710 TPLT; product number 81041) and Hamilton 30 gauge sharp-tipped needle with plastic hub (KF 730NDL 6/package, 30)G/2'; product number 90130) was used in the center of treatment injection for NP. For the vehicle group, 15 microliters of hydrogel was aspirated in a syringe and then slowly injected into each disc (injection in less than 4.5 minutes to facilitate liquid injection prior to gelation). For the cell + buffer (phosphate buffered saline solution) group, 15 microliters of 10 in sterile PBS was added5One cell was injected into each disc. For cells in the vehicle group, 10 microliters of hydrogel vehicle was injected5And (4) cells. The injection was performed under c-arm guidance to confirm that the needle was in the center of the disc. All injections were performed at a deliberately slow and constant rate over the course of one minute to avoid a rapid increase in disc pressure and subsequent expulsion of material. The rabbits were sutured, resuscitated, and cared for as described for the puncture surgery.
Magnetic resonance imaging
Sagittal MRIs were obtained at time 0 (before circular puncture), 3 weeks (before injection surgery), 6 weeks and 12 weeks (before sacrifice). T1-weighted images (TR 650ms, TE 14ms, layer thickness 0.6mm) and T2-weighted images (TR 3800ms, TE 114ms, layer thickness 0.6mm) were obtained using a3 tesla Siemens (Siemens) magnet and a standard human knee coil. The rabbits were sedated and placed in a supine position in a knee coil. The T1 weighted image is used to qualitatively check for any bone abnormalities in the spine. The T2 weighted image is used to quantify the amount of degeneration in the disc. The midsagittal layer of the T2 weighting sequence is determined by a trained physician based on the width of the vertebral bodies, spinal cord, and spinous processes. NPs that are regions of interest are determined using previously validated automated segmentation methods (see Bechara et al, am J neuroradiology.2010; 31(9): 1640-. The MRI index is calculated as the sum of the pixel areas of each pixel within the region of interest multiplied by the pixel intensity. The percent NP area and MRI index for the three discs of interest were averaged relative to the control value at week 0 and plotted against time points. Statistical significance was calculated using student T-test (p < 0.05).
Sacrifice and sample processing
All rabbits were sacrificed 12 weeks after initial puncture surgery (after obtaining the final MRI). Immediately after death, the entire spine was excised. Disc L4-5 was prepared for histological analysis. Fix and use the intervertebral disc DECALCIFIER(Surgipath Medical index, Inc.) decalcified for two weeks, then dehydrated in a histological tissue processor, and embedded in paraffin. Next, the disc was cut to a thickness of 5 μm in the sagittal plane. Sections were processed with hematoxylin-eosin (H) according to standard histological protocols&E) (sigma) stained and photographed using a Nikon (Nikon) E800 microscope.
Biomechanics of biology
After sacrifice, disc L3-4 was cut into functional spinal units (FSU, bone-disc-bone) for biomechanical analysis. The rear structure of The sample was removed, The sample was embedded in epoxy resin, and fitted with a custom fixture in an axial testing machine controlled with Matlab (MatlabR2008a, meiswoko corporation of midkey, MA Mathworks, inc. All discs were packed in saline soaked gauze to minimize dehydration and tested on the same day of sacrifice. The samples were pretreated with a compression load of 20 cycles (0 to 1.0MPa, 0.1mm/s) followed by constant compression (1.0MPa) for 1100 seconds. This loading target was chosen because it corresponds to the pressure experienced by the human IVD in activities of daily living (adjustment for the smaller size of the rabbit intervertebral disc) (Wilke et al, spine.1999; 24; 8): 755-. The initial ramp phase is defined as the first 200 microseconds of the test, while the creep phase is defined as the remainder of the test. After testing, the creep curve was fitted with the following two-phase exponential model:
wherein d (t) is the axial displacement over time, L0For an applied axial load, S1And S2Is an elastic damping coefficient (N/mm), and η1And η2Is a viscous damping coefficient (Ns/mm). An average curve was generated and fitted with an exponential model for each condition.
Results
Rabbit
No adverse effects from treatment were observed in any of the groups.
MRI
T1-weighted median sagittal plane MRI of the L2-3, L3-4, and L4-5 discs showed no significant change or osteophyte formation. T2-weighted mid-sagittal MRI degeneration (ROI darkened and collapsed from 0 to 12 weeks) of the discs in the punctured group compared to the unpunctured control. As shown in fig. 1 and 2, all three treatment groups qualitatively showed a smaller degree of degeneration than the puncture group. The punctured (and untreated) discs showed the greatest degree of degeneration with 59% reduction in NP area and 64% reduction in MRI index with increasing time points. All three treatment groups showed a smaller degree of regression compared to the puncture group based on total NP area and MRI index. The vehicle + cell group had the smallest area and MRI index decline among the treatment groups, and was most similar to the control group (fig. 3). At weeks 0 and 3, there was no statistically significant difference in MRI index or area between the control and any of the three treatment and puncture groups, or between the puncture and treatment groups. At 6 and 12 weeks, the MRI index and area differences between control and puncture groups were statistically significant, which was expected for an effective regression model. The control and vehicle groups showed statistically significant differences in MRI index at 6 and 12 weeks and statistically significant differences in area at 12 weeks. The MRI indices at 12 weeks for the control and buffer + cell groups were statistically significantly different. The MRI index and area at 6 and 12 weeks were statistically significantly different for both the control and vehicle + cell groups. The puncture and vehicle groups showed no statistically significant difference in MRI quantification with increasing time points. Both the MRI index and area at 6 and 12 weeks were statistically significantly different for the puncture and buffer + cell groups. The MRI index and area at 12 weeks were significantly different for the puncture group and the vehicle + cell group.
Biomechanics of biology
The curves representing the normalized displacement of the total load (initial ramp phase and subsequent creep phase) are given in fig. 4. There was a trend towards differences between the different groups. Specifically, the control group generated a curve similar to the vehicle + cell group, the puncture group similar to the buffer + cell group, and the vehicle group between these groups. Most of the variability between these groups comes from the initial ramp phase. Despite these trends, the early and late viscous and elastic damping coefficients did not produce any statistically significant differences when the creep phase of the curve was fitted with a two-phase exponential model (data not shown).
Histology
Representative sagittal sections of disc L4-5 were stained with hematoxylin-eosin (H & E) and viewed at 20X and 100X magnification (FIGS. 5,6 and 7). The control disc retains its architecture. It is predicted that the punctured disc shows loss of cellular structure and fibrosis in the NP, indicating degeneration. Punctured discs treated with hydrogel carriers result in some loss of cellular structure but retain a robust extracellular matrix. Treatment with cell + buffer showed relative retention of NP area, but significant fibrosis was observed. Treatment with cell + carrier results in improved cell structure and architecture compared to punctured discs.
Discussion of the related Art
This example encompasses a wide and diverse range of outcome indicators, including novel MRI techniques and biomechanics, so as to not only quantify disc degeneration (IDD), but also to be able to demonstrate responses to targeted treatment of disc degeneration. As discussed in detail in this example, the treated group exhibited different viscoelastic properties than the control and puncture group values.
Injection of rabbit lumbar disc (IVD) undergoing IDD with hUTC in vehicle had beneficial effects compared to vehicle alone or cell + buffer alone, according to MRI, histology and biomechanical criteria. All three treatments showed beneficial effects compared to untreated punctured discs. The Nucleus Pulposus (NP) area and index on MRI for all three treatment groups was between control (non-degenerative) and puncture group (maximally-degenerative) conditions, indicating that this treatment helped partially delay disc height and signal intensity changes. The axial load produces a displacement curve that appears different for each condition. Histology shows restoration of the process to make the disc architecture and cell structure variable.
The cell + PBS group produced MRI data that fell between the control and puncture group values and appeared very similar to the MRI data of the vehicle group. However, the displacement curves of this set most closely resemble the punctured state, suggesting that while this process helps to partially restore NP area and signal strength on MRI, it is not very effective in restoring the natural mechanical properties of the disc.
The individual carrier groups produced MRI data that fell between the values of the punctured and unpunctured groups. In this example, the displacement curve of the hydrogel set also fell between the punctured and unpunctured states, indicating that the hydrogel carrier was able to restore some of the mechanical properties of the disc regardless of any extracellular matrix synthesis of native or transplanted cells. The tissue structure of the vehicle group had more cell structure than expected from the non-cell treatment, possibly indicating that the hydrogel provided a safe harbor where native cells could thrive.
The cell + carrier group combines cell transplantation and hydrogel injection of the other two groups. This group had a consistent but not significant trend towards better MRI degeneration indicators than the other two treatment groups (most similar to the control state, but still significantly different from the control values). The set also had a displacement curve most similar to the control curve, indicating the best mechanical response. The tissue structure of this group had significantly increased cellular structure and relatively preserved architecture compared to the punctured group, but there was evidence of fibrosis.
There were no histological indications that transplantation of human cells into rabbit intervertebral discs would produce an immune response. None of the treated rabbits showed signs of illness and no evidence of a histological inflammatory response.
Human postpartum umbilical cord tissue is an attractive source of therapeutic cells because donor tissue is readily available and cells are easily harvested, while there are no ethical conflicts associated with embryonic stem cells or fetal cells.
In summary, it can be predicted that MRI and histological signs of degeneration occurred in the punctured group compared to the unpunctured control. The treatment group developed a lower degree of MRI and histological signs of degeneration compared to the puncture group. The treated group showed different viscoelasticity values than the control and puncture group values. This study showed that injecting rabbit waist IVD undergoing degeneration with (a) hUTC and (b) hydrogel slowed the process of IVD degeneration. Cells injected into the hydrogel slowed the progression of degeneration to a greater extent than either (a) hUTC alone or (b) hydrogel alone. Thus, the study showed that treatment of punctured rabbit intervertebral discs with human umbilical cord tissue-derived cells (hUTC) with or without a hydrogel carrier solution helped to restore MRI, histological and biomechanical properties to values close to those of the unpunctured controls.
<110> Depuyinsts products Ltd
<120> treatment of intervertebral disc degeneration using human umbilical cord tissue-derived cells and hydrogel
<130>18668-332000 (0244C1WO)
<140>
<141>2012-5-18
<150>13/111,933
<151>2011-5-19
<150>12/337,439
<151>2008-12-17
<150>61/016,849
<151>2007-12-27
<160>10
<170> PatentIn version 3.5
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