阅读说明：本技术 诱导性多能干细胞及其制备方法 (induced pluripotent stem cell and preparation method thereof ) 是由 裴雪涛 裴海云 岳�文 李慧琳 聂纪芹 曲洺逸 范增 张博文 贾雅丽 何丽娟 南 于 2018-05-22 设计创作，主要内容包括：本发明提供了诱导性多能干细胞,所述诱导性多能干细胞来源于脐动脉内皮细胞且可以分化为生血内皮细胞。本发明的诱导性多能干细胞保留了部分脐动脉内皮细胞的表观遗传特性,能够分化为血液系统和内皮系统的共祖细胞—生血内皮细胞。由此为造血干细胞移植以及进一步诱导分化获得充足数量的红细胞或血小板乃至各种类型的血液细胞提供来源,也为心血管相关疾病涉及的血管新生提供充足的、具有良好功能的内皮干/祖细胞或成熟的内皮种子细胞,具有较高的应用价值。(The present invention provides induced pluripotent stem cells that are derived from umbilical artery endothelial cells and can differentiate into hematopoietic endothelial cells. The induced pluripotent stem cells of the invention retain the epigenetic characteristics of a part of umbilical artery endothelial cells and can be differentiated into the hematopoietic endothelial cells which are the co-progenitors of the blood system and the endothelial system. Therefore, the method provides a source for obtaining sufficient red blood cells or platelets and various types of blood cells for hematopoietic stem cell transplantation and further induced differentiation, also provides sufficient endothelial stem/progenitor cells or mature endothelial seed cells with good functions for angiogenesis related to cardiovascular diseases, and has high application value.)

1. An induced pluripotent stem cell derived from umbilical artery endothelial cells and capable of differentiating into hematopoietic endothelial cells.

2. the induced pluripotent stem cell of claim 1, wherein the induced pluripotent stem cell is non-genomically integrated;

optionally, the induced pluripotent stem cells are obtained by reprogramming the umbilical artery endothelial cells;

Optionally, the reprogramming comprises transfecting sendai virus carrying reprogramming factors into the umbilical artery endothelial cells;

Optionally, the induced pluripotent stem cell expresses a TIE2 gene;

Optionally, genes of at least one of the following are down-regulated in expression levels within the induced pluripotent stem cells as compared to the umbilical artery endothelial cells: CD31 gene, CD144 gene, vWF gene and TIE1 gene;

optionally, the hematogenic endothelial cells can differentiate into cells of at least one of: blood lineage cells, endothelial stem/progenitor cells, endothelial cells, and vascular smooth muscle cells;

Optionally, the hematopoietic endothelial cells are induced to differentiate into hematopoietic stem cells and further into hematopoietic lineage cells, wherein CD43 is a cell population resulting from the induced differentiation of the hematopoietic endothelial cells into the hematopoietic lineage cells⁺The cell content is 35-50%, and the CD45⁺the cell content is 15-20%.

3. A medicament comprising umbilical artery endothelial cells or induced pluripotent stem cells according to claim 1 or 2.

4. Use of an induced pluripotent stem cell according to claim 1 or claim 2 in the preparation of a medicament for the treatment of a cardiovascular related disease.

5. A method of screening for an agent comprising a TIE2 kinase inhibitor or a TIE2 kinase agonist comprising:

Contacting a candidate agent with the induced pluripotent stem cell according to claim 1 or 2, and determining the expression level of TIE2 gene in the induced pluripotent stem cell before and after the contacting,

down-regulation of the expression of the TIE2 gene is indicative of the candidate agent being an agent comprising a TIE2 kinase inhibitor;

The TIE2 gene expression being upregulated is indicative of the candidate agent as an agent comprising a TIE2 kinase agonist.

6. A kit comprising the induced pluripotent stem cell according to claim 1 or 2.

7. A method for obtaining an induced pluripotent stem cell according to claim 1 or claim 2, comprising:

Reprogramming the umbilical artery endothelial cells to obtain the induced pluripotent stem cells;

Optionally, the step of obtaining the umbilical artery endothelial cells comprises:

Incubating umbilical artery blood vessels filled with type IV collagenase solution, and collecting umbilical artery endothelial cells in the obtained digestive juice;

optionally, the concentration of the type IV collagenase solution is 0.5-3 mg/mL, and the incubation time is 10-20 minutes;

Optionally, the steps include:

removing residual blood in umbilical artery blood vessels of an umbilical cord, and filling 1mg/mL type IV collagenase solution into the obtained blood vessels;

Sealing both ends of the vessel at CO₂Incubating for 15 minutes at 37 ℃ in the atmosphere, and collecting digestive juice in the blood vessel;

Centrifuging the digestive juice, and discardingCleaning, suspending cells by EGM-2 medium, and suspending the obtained cell suspension according to the proportion of 1-5 × 10⁵Individual cells/well into well plate in CO₂Incubating at 37 ℃ in the atmosphere, replacing the culture medium every other day, washing the cells once by PBS when the cells grow to 80-90% of the cells are confluent, digesting at 37 ℃ for 3-5min by 0.25% tryptin-EDTA, blowing the cells from the culture medium after terminating the digestion by EGM-2 culture medium, collecting the cells in a 15ml centrifuge tube, centrifuging at the room temperature at the rotation speed of 1000rpm for 5min, and removing the supernatant so as to obtain the umbilical artery endothelial cells;

Optionally, the reprogramming comprises transfecting sendai virus carrying reprogramming factors into the umbilical artery endothelial cells.

8. a hematopoietic endothelial cell obtained by differentiation of the induced pluripotent stem cell according to claim 1 or 2.

9. a method of obtaining the hematopoietic endothelial cells of claim 8, comprising:

pre-culturing the induced pluripotent stem cells;

differentiating the pre-cultured cells to obtain mesodermal cells;

And differentiating the mesoderm cells to obtain the hematogenous endothelial cells.

10. A hematopoietic stem cell obtained by differentiation of the hematopoietic endothelial cell according to claim 8.

Technical Field

The present invention relates to the field of biology. In particular, the invention relates to induced pluripotent stem cells and methods of making the same. More specifically, the invention relates to induced pluripotent stem cells, a medicament, use of induced pluripotent stem cells in the preparation of a medicament, a method of screening a preparation, a kit, a method of obtaining induced pluripotent stem cells, hematopoietic endothelial cells, a method of obtaining hematopoietic endothelial cells, and hematopoietic stem cells.

Background

hematopoietic Stem Cells (HSCs) have a high self-renewal capacity and a multipotentiality and can produce all types of blood cells, such as erythrocytes, leukocytes, platelets, lymphocytes, etc. It not only can rebuild the whole hemopoietic system in the life process, but also has the function of maintaining long-term hemopoiesis. In recent years, HSC transplantation is increasingly applied to clinical treatment of malignant tumors of blood or non-blood systems, showing wide prospects, but the shortage of absolute number greatly limits the application development. Since the mechanism of hematopoietic stem cell self-renewal and maintenance is not clear, it is difficult to achieve expansion culture in vitro, and the regeneration of HSC from outside of the body becomes another way to solve the problem of insufficient cell number for clinical transplantation.

Somatic cell reprogramming can be achieved in mice, humans, rats, monkeys, and dogs by reprogramming somatic cells into induced pluripotent stem cells (iPS cells) by exogenously expressing several genes. The iPS cell has great potential value in the aspects of specific cell transplantation treatment of patients, research on pathogenesis and new drug screening.

Originally iPS cells were established by the transport of transgenes via Moloney Murine Leukemia Virus (MMLV) -based retroviral vectors. Retroviruses stably infect mouse fibroblasts and mediate their RNA genome into the genome of the host by means of reverse transcriptase. Therefore, a large number of transgenes are integrated into the genome of the iPS cell, thereby achieving sustained expression.

iPS cells were initially established from primary cultured mouse fibroblasts, and were subsequently reprogrammed in multiple species, multiple different types of tissue cells. Epigenetic alterations are one of the manifestations of the formation of iPS cells. Although both iPS and ES cells belong to pluripotent cells, they have similar gene expression profiles. However, they differ in apparent modification, particularly those genes that do not involve pluripotency. Cell differentiation is a process of limiting differentiation potential through epigenetic modification, each type of somatic cell having specific epigenetic characteristics to stabilize itself. Exogenous expression of reprogramming factors can affect several downstream genes in somatic cells and alter their apparent modifications. However, it is difficult to imagine that these several factors can control all genes of the genome. Indeed, whole genome analysis showed that iPS and ES cells have similar DNA methylation patterns. Uncontrolled genes will still retain their epigenetic state, which will affect the differentiation potential of iPS cells. For example, the methylation status of the enhancer binding site of the GFAP gene of astrocytes controls the differentiation fate of neural progenitor cells by altering the binding activity to the enhancer STAT 3. They tend to become astrocytes when unmethylated and they tend to differentiate neuronally when methylated. It can be seen that epigenetic memory is an important factor affecting the differentiation propensity of iPS.

currently, methods for generating HSCs in vitro using iPS cells remain to be investigated.

disclosure of Invention

the present invention aims to solve at least to some extent at least one of the technical problems of the prior art.

To this end, in one aspect of the invention, the invention features an induced pluripotent stem cell. According to an embodiment of the invention, the induced pluripotent stem cells are derived from umbilical artery endothelial cells and can differentiate into hematogenic endothelial cells. The inventor takes umbilical artery endothelial cells as starting cells, and can obtain induced pluripotent stem cells after treatment (such as reprogramming) and the cells can be further differentiated into hematogenic endothelial cells. Since hematopoietic endothelial cells are co-progenitors of hematopoietic stem cells and endothelial cells, hematopoietic stem cells and endothelial cells can be obtained by differentiating hematopoietic endothelial cells, thereby providing a source for obtaining a sufficient number of red blood cells or platelets and even various types of blood cells for hematopoietic stem cell transplantation and further induced differentiation. In addition, endothelial stem/progenitor cells, endothelial cells or vascular smooth muscle cells, which can be differentiated from hematopoietic endothelial cells, can also provide sufficient and well-functioning seed cells for angiogenesis involved in cardiovascular-related diseases (e.g., common treatment of ischemic diseases of the lower extremities).

According to embodiments of the present invention, the induced pluripotent stem cell may further have the following additional technical features:

according to embodiments of the invention, the induced pluripotent stem cell is non-genomically integrated.

According to an embodiment of the invention, the induced pluripotent stem cells are obtained by reprogramming the umbilical artery endothelial cells.

according to an embodiment of the present invention, the reprogramming comprises transfecting Sendai virus carrying reprogramming factors into the umbilical artery endothelial cells.

According to an embodiment of the invention, the induced pluripotent stem cell expresses the TIE2 gene.

according to an embodiment of the invention, the expression level of at least one of the following genes is down-regulated in the induced pluripotent stem cells compared to the umbilical artery endothelial cells: CD31 gene, CD144 gene, vWF gene and TIE1 gene.

According to an embodiment of the invention, the hematogenic endothelial cells can differentiate into cells of at least one of: blood lineage cells, endothelial stem/progenitor cells, endothelial cells, and vascular smooth muscle cells.

According to the embodiment of the present invention, the hematopoietic endothelial cells can be induced to differentiate into hematopoietic stem cells and further into hematopoietic lineage cells, wherein CD43 is a cell population obtained by inducing differentiation from the hematopoietic endothelial cells into the hematopoietic lineage cells⁺The cell content is 35-50%, and the CD45⁺The cell content is 15-20%.

in another aspect of the invention, a medicament is provided. According to an embodiment of the invention, the medicament comprises umbilical artery endothelial cells or induced pluripotent stem cells as described above.

in a further aspect of the invention, the invention provides the use of an induced pluripotent stem cell as hereinbefore described in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment of cardiovascular related diseases.

In yet another aspect, the invention features a method of screening for a drug comprising a TIE2 kinase inhibitor or a TIE2 kinase agonist. According to an embodiment of the invention, the method comprises: contacting a candidate preparation with said induced pluripotent stem cell, and determining the amount of expression of the TIE2 gene in said induced pluripotent stem cell before and after the contacting, wherein downregulation of the TIE2 gene expression is indicative of the candidate preparation as a preparation comprising a TIE2 kinase inhibitor; the TIE2 gene expression being upregulated is indicative of the candidate agent as an agent comprising a TIE2 kinase agonist.

In yet another aspect of the invention, the invention provides a method for obtaining an induced pluripotent stem cell as described above. According to an embodiment of the invention, the method comprises: reprogramming the umbilical artery endothelial cells to obtain the induced pluripotent stem cells.

according to an embodiment of the present invention, the step of obtaining the umbilical artery endothelial cells comprises: and (3) incubating umbilical artery blood vessels filled with the collagenase type IV solution, and collecting umbilical artery endothelial cells in the obtained digestive juice.

According to the embodiment of the invention, the concentration of the collagenase type IV solution is 0.5-3 mg/mL, and the incubation time is 10-20 minutes.

According to an embodiment of the invention, the steps comprise: removing residual blood in umbilical artery blood vessels of an umbilical cord, and filling 1mg/mL type IV collagenase solution into the obtained blood vessels; sealing both ends of the vessel at CO₂Incubating for 15 minutes at 37 ℃ in the atmosphere, and collecting digestive juice in the blood vessel; centrifuging the digestive juice, removing supernatant, suspending cells by EGM-2 medium, and suspending the obtained cell suspension according to the proportion of 1-5 multiplied by 10⁵individual cells/well into well plate in CO₂Incubating at 37 ℃ in atmosphere, replacing culture medium every other day, washing cells once by PBS when the cells grow to 80-90% of cells are confluent, digesting for 3-5min at 37 ℃ by 0.25% tryptin-EDTA, blowing the cells from the culture medium after terminating digestion by EGM-2 culture medium, collecting the cells in a 15ml centrifuge tube, centrifuging for 5min at the room temperature at the rotation speed of 1000rpm, and removing supernatant so as to obtain the umbilical artery endothelial cells.

according to an embodiment of the present invention, the reprogramming comprises transfecting Sendai virus carrying reprogramming factors into the umbilical artery endothelial cells.

in yet another aspect of the invention, the invention features a hematogenic endothelial cell. According to an embodiment of the present invention, the hematopoietic endothelial cells are differentiated from the induced pluripotent stem cells as described above.

In yet another aspect of the present invention, the present invention provides a hematopoietic stem cell. According to an embodiment of the present invention, said hematopoietic stem cells are obtained by differentiation of hematopoietic endothelial cells as described above.

In yet another aspect of the invention, the invention provides a method of obtaining the aforementioned hematogenic endothelial cells. According to an embodiment of the invention, the method comprises: pre-culturing the induced pluripotent stem cells; differentiating the pre-cultured cells to obtain mesodermal cells; and differentiating the mesoderm cells to obtain the hematogenous endothelial cells.

In yet another aspect of the invention, the invention features a kit. According to an embodiment of the invention, the kit comprises induced pluripotent stem cells as described above.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

drawings

the above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a light mirror image of primary cultured umbilical artery endothelial cells according to one embodiment of the present invention;

FIG. 2 shows a schematic representation of a surface marker analysis for flow cytometry detection of umbilical artery endothelial cells according to one embodiment of the present invention;

FIG. 3 shows an immunofluorescence map of vWF expression in umbilical artery endothelial cells according to one embodiment of the present invention;

FIG. 4 shows a light mirror view of the lumen of the umbilical artery endothelial cells according to one embodiment of the present invention;

FIG. 5 shows a light mirror image of clone formation during reprogramming according to one embodiment of the present invention, where A: starting cell morphology, B: appearance of iPS clones, C: cloning iPS to culture cells in a feeder-free environment;

FIG. 6 shows A schematic diagram of an analysis of the expression of the pluripotency markers SSEA-4 and TrA-1-60 in iPS-A by flow-assay according to one embodiment of the invention;

FIG. 7 shows A schematic analysis of qPCR analysis of endogenous pluripotency and endothelial-specific gene expression in iPS-A according to one embodiment of the invention;

FIG. 8 shows a schematic view of karyotyping according to an embodiment of the present invention;

FIG. 9 shows A schematic diagram of an identification analysis of the in vitro differentiation potential of iPS-A according to one embodiment of the present invention; and

FIG. 10A shows A schematic cell morphology of iPS-A and embryonic stem cell differentiation into hematopoietic lineage cells according to one embodiment of the present invention;

FIG. 10B shows a schematic representation of the analysis of the expression of flow detection markers CD43 and CD45, according to one embodiment of the present invention;

FIG. 10C shows a schematic of an analysis of hematopoietic associated gene expression according to one embodiment of the invention; and

FIG. 10D shows A schematic of an analysis of endothelial-associated gene expression after differentiation of iPS-A into hematogenic endothelium, according to one embodiment of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The invention provides an induced pluripotent stem cell, a medicine, application of the induced pluripotent stem cell in preparation of the medicine, a method for screening a preparation, a kit, a method for obtaining the induced pluripotent stem cell, a hematogenic endothelial cell, a method for obtaining the hematogenic endothelial cell and a hematopoietic stem cell, which are respectively described in detail below.

induced pluripotent stem cells

In one aspect of the invention, the invention features an induced pluripotent stem cell. According to an embodiment of the invention, the induced pluripotent stem cells are derived from umbilical artery endothelial cells and can differentiate into hematogenic endothelial cells.

it was found that hematopoietic stem cells appear in large arteries, one of which is the umbilical artery, after the allantois and chorion are fused into a placenta, and subsequent studies have further confirmed that endothelial cells and hematopoietic stem cells share a common ancestral cell, the hematogenic endothelium.

in view of the above, the inventors have obtained induced pluripotent stem cells by treating (e.g., reprogramming) umbilical artery endothelial cells as starting cells, which have high self-replication ability and multipotentiality and can provide sufficient cell sources. Compared with the existing embryonic stem cells or induced pluripotent stem cells, the cells are derived from umbilical artery endothelial cells, and the epigenetic characteristics of the starting cells are likely to be partially preserved after being reprogrammed into the induced pluripotent stem cells, so that the cells are likely to contribute to the further differentiation of the induced pluripotent stem cells into hematogenous endothelium and even cell types of hematopoietic lineages and endothelial lineages, can be used as novel source cells of hematogenous endothelial cells, hematopoietic stem cells and endothelial progenitor cells, and provide a source for obtaining sufficient quantities of red blood cells or blood platelets and even various types of blood cells for hematopoietic stem cell transplantation and further induced differentiation. In addition, endothelial stem/progenitor cells, endothelial cells or vascular smooth muscle cells differentiated from hematopoietic endothelial cells can also provide sufficient, well-functioning seed cells for angiogenesis involved in cardiovascular-related diseases (e.g., common treatment of ischemic diseases of the lower extremities).

According to embodiments of the invention, the induced pluripotent stem cell is non-genomically integrated. The target cell is transfected with a retrovirus carrying the target gene to thereby mediate the target gene into the genome of the target cell. It has been found that in both ES cells and induced pluripotent stem cells, DNA methylation of the retroviral promoter region is inactivated, and therefore expression of a target gene carried by a retrovirus is gradually suppressed during reprogramming, and the target gene is completely silenced when the cells become true induced pluripotent stem cells. It is this auto-silencing mechanism that is thought to achieve efficient somatic reprogramming. However, the exogenous sequence remains in the genome of the iPS cell and alterations in the protostructure may induce some abnormalities. In particular the proto-oncogene c-Myc in reprogramming factors, whose reactivation may lead to transgene-derived tumor formation. To prepare safe iPS cells, the inventors used a non-genomic integration method to reprogram, thereby obtaining non-genomic integrated induced pluripotent stem cells.

according to an embodiment of the invention, the induced pluripotent stem cells are obtained by reprogramming umbilical artery endothelial cells. The inventors have found that by using umbilical artery endothelial cells as starting cells, induced pluripotent stem cells can be obtained by treatment (e.g., reprogramming), and that the induced pluripotent stem cells partially retain the epigenetic characteristics of the starting cells, thereby facilitating further differentiation of the induced pluripotent stem cells into hematopoietic endothelium and even cell types of hematopoietic and endothelial lineages.

According to an embodiment of the present invention, reprogramming comprises transfecting Sendai virus carrying reprogramming factors into umbilical artery endothelial cells. According to a particular embodiment of the invention, the reprogramming factors are selected from Oct3/4, Sox2, Klf4, and c-Myc. By transfecting a retrovirus carrying a reprogramming factor into a host cell (umbilical artery endothelial cell), the reprogramming factor is capable of inducing the overexpression of numerous target genes, many of which are likely to be involved in the production of induced pluripotent stem cells. Furthermore, the inventors found that Sendai virus, which is a single negative strand RNA virus, has a high safety because the life cycle is completely carried out in the cytoplasm and is not integrated into the genome, as compared with other retroviruses. And the second generation Sendai virus contains temperature sensitive mutation, and can remove the carrier remained in the coating slurry after transfection by changing the culture temperature, thereby ensuring the application safety. According to a specific example of the present invention, the second generation Sendai virus was purchased from Invitrogen corporation.

The term "reprogramming" as used herein means that adult cells form different types of cells including stem cells by changing their differentiation state.

According to an embodiment of the present invention, the induced pluripotent stem cell expresses the TIE2 gene. The TIE2 gene encodes the pro-angiogenic protein factor TIE2, which is expressed primarily in hematopoietic and vascular endothelial cells. Because the induced pluripotent stem cell is derived from the umbilical artery endothelial cell, the TIE2 gene expression of the umbilical artery endothelial cell is kept. However, TIE2 gene was not expressed in induced pluripotent stem cells and ES cells from other sources.

According to an embodiment of the present invention, the expression level of at least one of the following genes is down-regulated in induced pluripotent stem cells compared to umbilical artery endothelial cells: CD31 gene, CD144 gene, vWF gene and TIE1 gene.

according to an embodiment of the invention, the hematopoietic endothelial cells can differentiate into cells of at least one of: blood lineage cells, endothelial stem/progenitor cells, endothelial cells, and vascular smooth muscle cells. It is found that endothelial cells and hematopoietic stem cells have the same ancestral cells, namely hematopoietic endothelium, because the hematopoietic endothelial cells are differentiated from induced pluripotent stem cells derived from umbilical artery endothelial cells, and the induced pluripotent stem cells partially retain the epigenetic characteristics of the umbilical artery endothelial cells, so that the endothelial cells and the hematopoietic progenitor cells, namely hematopoietic endothelial cells, are more prone to be differentiated into endothelial lineage cells or hematopoietic stem cells and various types of blood lineage cells downstream of the hematopoietic stem cells respectively after induction. In accordance with an embodiment of the present invention, CD43 is a cell population obtained by inducing differentiation of hematopoietic endothelial cells into blood lineage cells⁺(blood system cell marker appearing earliest in development process) cell content is 35-50%, CD45⁺(the marker expressed by all blood system cells except the enucleated mature erythrocytes) is 15-20%. Therefore, the blood line cell yield obtained by carrying out a series of differentiation on the induced pluripotent stem cells derived from the umbilical artery endothelial cells is higher.

It should be noted that the term "blood cells" used herein may also be referred to as blood cells, and should be understood in a broad sense, that is, all cells present in blood can be regarded as blood cells, including but not limited to red blood cells, white blood cells, platelets, lymphocytes, and the like.

Medicine

in another aspect of the invention, a medicament is provided. According to an embodiment of the invention, the medicament comprises umbilical artery endothelial cells or induced pluripotent stem cells as described above. The induced pluripotent stem cells are derived from umbilical artery endothelial cells, and the epigenetic characteristics of the umbilical artery endothelial cells are partially reserved, so that the induced pluripotent stem cells can be further differentiated into hematopoietic endothelial cells and cell types of hematopoietic lineages, can be used as novel source cells of the hematopoietic endothelial cells, the hematopoietic stem cells and the endothelial progenitor cells, and provide sources for hematopoietic stem cell transplantation and further induced differentiation to obtain sufficient red blood cells or platelets and various types of blood cells, and in addition, the endothelial stem/progenitor cells, the endothelial cells or the vascular smooth muscle cells obtained by differentiation of the hematopoietic endothelial cells can also provide sufficient seed cells with good functions for angiogenesis related diseases (such as common treatment of lower limb ischemic diseases).

the medicaments of the invention may be used in conjunction with conventional methods of treatment and/or therapy, or may be used separately from conventional methods of treatment and/or therapy. For example, it may be administered according to any conventional method in the art, together with other stem cells for transplantation and other purposes, drugs, or in admixture therewith. When the agents of the present invention are administered in combination therapy with other agents, they may be administered to the individual sequentially or simultaneously. Alternatively, the medicament of the invention may comprise a combination of the umbilical artery endothelial cells or induced pluripotent stem cells of the invention, a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, and other therapeutic or prophylactic agents known in the art.

pharmaceutically acceptable carriers or pharmaceutically acceptable excipients include: lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, analgesic control agents, solubilizers, stabilizers, and preservatives. Details of pharmaceutically acceptable carriers and excipients can be found in Remington's Pharmaceutical Sciences (19the d., 1995), which is incorporated herein by reference.

It will be appreciated by those skilled in the art that the features and advantages described above for induced pluripotent stem cells apply equally to this drug and will not be described in further detail herein.

Use of

In a further aspect of the invention, the invention provides the use of an induced pluripotent stem cell as hereinbefore described in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment of cardiovascular related diseases. The induced pluripotent stem cells are derived from umbilical artery endothelial cells, and the epigenetic characteristics of the umbilical artery endothelial cells are partially reserved, so the induced pluripotent stem cells can be further differentiated into the cell types of hematogenous endothelium and even hematopoietic lineages, can be used as novel source cells of the hematogenous endothelial cells, hematopoietic stem cells and endothelial progenitor cells, provide sources for obtaining sufficient red blood cells or platelets and even various types of blood cells for hematopoietic stem cell transplantation and further induced differentiation, and also provide sufficient seed cells with good functions for angiogenesis related to cardiovascular related diseases.

The term "treatment" as used herein is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease (e.g., preventing cardiovascular-related disease) or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug containing a compound described herein to an individual in need thereof.

It will be appreciated by those skilled in the art that the features and advantages described above for induced pluripotent stem cells apply equally to this use and will not be described in further detail herein.

Method for screening preparation

In yet another aspect, the invention features a method of screening for an agent that includes a TIE2 kinase inhibitor or a TIE2 kinase agonist. According to an embodiment of the invention, the method comprises: contacting the candidate preparation with the induced pluripotent stem cells described above, and determining the expression level of the TIE2 gene in the induced pluripotent stem cells before and after the contacting, wherein the down-regulation of the TIE2 gene expression is indicative of the candidate preparation as a preparation comprising a TIE2 kinase inhibitor; upregulation of TIE2 gene expression is indicative of a candidate agent as an agent comprising a TIE2 kinase agonist.

Currently, common TIE 2-expressing cells such as endothelial cells are used primarily to study the kinase signaling pathway of the angiogenesis promoting protein factor (TIE2) to obtain TIE2 kinase inhibitors and agonists. However, the current cells expressing TIE2, such as endothelial cells, have the following problems: the separation and acquisition are complicated; the proliferation capacity is limited, and after several generations, the phenomena of function and characteristic loss and apoptosis can occur, and frequent repeated preparation is needed; each preparation lot will vary and the karyotype is not necessarily normal. In view of this, the inventors have found that induced pluripotent stem cells obtained by reprogramming umbilical artery endothelial cells retain the expression of the TIE2 gene in umbilical artery endothelial cells, and thus, it is possible to obtain agonists (activation pathway, up-regulation of TIE2 gene expression) or inhibitors (inhibition pathway, down-regulation of TIE2 gene expression) of TIE2 kinase by studying the effect of candidate agents on the expression pathway of TIE2 kinase (encoded by TIE2 gene).

Compared with other TIE 2-expressing cells, the induced pluripotent stem cells have high self-renewal capacity and amplification capacity, can be subjected to unlimited passage, cryopreservation and resuscitation, and maintain stable and normal karyotypes. In addition, the medicine can be derived from the self body, so that one-to-one disease treatment or medicine screening is realized, and the rejection phenomenon is not easy to occur.

It will be appreciated by those skilled in the art that the features and advantages described above for induced pluripotent stem cells apply equally to the method of screening for agents and will not be described in detail here.

method for obtaining induced pluripotent stem cells

In yet another aspect of the invention, the invention provides a method for obtaining an induced pluripotent stem cell as described above. According to an embodiment of the invention, the method comprises: the umbilical artery endothelial cells are reprogrammed to obtain induced pluripotent stem cells. The inventors have found that by using umbilical artery endothelial cells as starting cells, induced pluripotent stem cells can be obtained by treatment (e.g., reprogramming), and that the induced pluripotent stem cells partially retain the epigenetic characteristics of the starting cells, thereby facilitating further differentiation of the induced pluripotent stem cells into hematopoietic endothelium and even into cell types of hematopoietic or endothelial lineage.

According to an embodiment of the present invention, the step of obtaining umbilical artery endothelial cells comprises: and (3) incubating umbilical artery blood vessels filled with the collagenase type IV solution, and collecting umbilical artery endothelial cells in the obtained digestive juice. The inventor finds that the type IV collagenase has mild action, can reduce the damage to cells in the process of digesting the cells, and has higher cell yield.

According to the embodiment of the invention, the concentration of the collagenase type IV solution is 0.5-3 mg/mL, and the incubation time is 10-20 minutes. The inventor obtains the better enzymolysis reaction condition through a large number of experiments, under the condition, the endothelial cells can fall off from the vessel wall, and simultaneously, the vascular smooth muscle cells adjacent to the next layer still remain on the vessel wall and do not fall off, thereby ensuring that the separated and obtained cells have high purity.

According to an embodiment of the invention, the step comprises: removing residual blood in umbilical artery blood vessels of an umbilical cord, and filling 1mg/mL type IV collagenase solution into the obtained blood vessels; sealing both ends of the vessel at CO₂Incubating for 15 minutes at 37 ℃ in the atmosphere, and collecting digestive juice in the blood vessel; centrifuging the digestive juice, removing supernatant, suspending cells by EGM-2 medium, and suspending the obtained cell suspension according to the proportion of 1-5 multiplied by 10⁵The individual cells/wells are accessed into a well plate,in CO₂Incubating at 37 ℃ in atmosphere, replacing the culture medium every other day, washing the cells once by PBS when 80-90% of the cells are confluent, digesting at 37 ℃ for 3-5min by 0.25% tryptin-EDTA, blowing the cells from the culture medium after terminating the digestion by EGM-2 culture medium, collecting the cells in a 15ml centrifuge tube, centrifuging at the room temperature at the rotation speed of 1000rpm for 5min, and removing the supernatant so as to obtain the umbilical artery endothelial cells. Therefore, the obtained umbilical artery endothelial cells have high yield and good activity.

According to an embodiment of the present invention, reprogramming comprises transfecting Sendai virus carrying reprogramming factors into umbilical artery endothelial cells. According to a particular embodiment of the invention, the reprogramming factors are selected from Oct3/4, Sox2, Klf4, and c-Myc. By transfecting a retrovirus carrying a reprogramming factor into a host cell (umbilical artery endothelial cell), the reprogramming factor is capable of inducing the overexpression of numerous target genes, many of which are likely to be involved in the production of induced pluripotent stem cells. Furthermore, the inventors found that Sendai virus, which is a single negative strand RNA virus, has a high safety because the life cycle is completely carried out in the cytoplasm and is not integrated into the genome, as compared with other retroviruses. And the second generation Sendai virus vector contains temperature sensitive mutation, and can remove the vector remained in the coating slurry after transfection by changing the culture temperature, thereby ensuring the application safety.

It will be appreciated by those skilled in the art that the features and advantages described above for induced pluripotent stem cells apply equally to the method of obtaining induced pluripotent stem cells and will not be described in detail here.

hematopoietic endothelial cells

In yet another aspect of the invention, the invention features a hematogenic endothelial cell. According to an embodiment of the present invention, the hematopoietic endothelial cells are differentiated from the induced pluripotent stem cells as described above. Because the induced pluripotent stem cells are derived from the human umbilical artery endothelial cells, the epigenetic characteristics of the human umbilical artery endothelial cells are partially preserved, so that the iPS cells tend to differentiate towards hematopoietic endothelial cells.

It will be appreciated by those skilled in the art that the features and advantages described above for induced pluripotent stem cells apply equally to the hematopoietic endothelial cells and will not be described in detail here.

Method for obtaining hematogenic endothelial cells

in yet another aspect of the invention, the invention provides a method of obtaining the aforementioned hematogenic endothelial cells. According to an embodiment of the invention, the method comprises: pre-culturing the induced pluripotent stem cells; differentiating the pre-cultured cells to obtain mesoderm cells; and differentiating the mesoderm cells to obtain the hematogenous endothelial cells. Therefore, the hematopoietic endothelial cells obtained by the method have high yield and good activity.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the hematopoietic endothelial cells apply equally to the method of obtaining hematopoietic endothelial cells and will not be described in detail here.

Hematopoietic stem cells

In yet another aspect of the present invention, the present invention provides a hematopoietic stem cell. According to an embodiment of the present invention, hematopoietic stem cells are obtained by differentiation of hematopoietic endothelial cells as described above. The research finds that endothelial cells and hematopoietic stem cells have the same ancestor cell, namely hematopoietic endothelium, and the hematopoietic endothelial cells are differentiated from induced pluripotent stem cells derived from umbilical artery endothelial cells, and the induced pluripotent stem cells partially retain the epigenetic characteristics of the umbilical artery endothelial cells, so that the induced pluripotent stem cells tend to be differentiated into the hematopoietic endothelial cells, and can be further differentiated into endothelial lineage cells or hematopoietic stem cells and downstream blood cells through specific induction.

It will be appreciated by those skilled in the art that the features and advantages described above for hematopoietic endothelial cells apply equally to the hematopoietic stem cells and will not be described in detail here.

Reagent kit

In yet another aspect of the invention, the invention features a kit. According to an embodiment of the invention, the kit comprises induced pluripotent stem cells as described above. Therefore, the kit of the invention can further obtain hematogenous endothelial cells, hematopoietic stem cells and endothelial cells, thereby providing a source for obtaining sufficient quantity of red blood cells or platelets and various types of blood cells for hematopoietic stem cell transplantation and further induced differentiation. In addition, endothelial stem/progenitor cells, endothelial cells or vascular smooth muscle cells, which can be differentiated from hematopoietic endothelial cells, can also provide sufficient and well-functioning seed cells for angiogenesis involved in cardiovascular-related diseases (e.g., common treatment of ischemic diseases of the lower extremities).

It will be appreciated by those skilled in the art that the features and advantages described above for induced pluripotent stem cells apply equally to the kit and will not be described further herein.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.