Compositions and methods for producing megakaryocytes
阅读说明：本技术 用于产生巨核细胞的组合物和方法 (Compositions and methods for producing megakaryocytes ) 是由 J·索恩 B·戴克斯特拉 于 2019-01-05 设计创作，主要内容包括：提供了由干细胞产生巨核细胞祖细胞(前MK)和巨核细胞(MK)的方法。本公开进一步提供了包含前MK和MK及其裂解物的组合物,以及使用前MK、MK、其裂解物及其组合物的方法。(Methods of producing megakaryocyte progenitor cells (pre-MKs) and Megakaryocytes (MKs) from stem cells are provided. The disclosure further provides compositions comprising pre-MK and lysates thereof, and methods of using pre-MK, lysates thereof, and compositions thereof.)
1. A method for producing megakaryocytes, comprising:
expanding pluripotent stem cells under low-adhesion or non-adhesion conditions and with agitation, wherein the expanded pluripotent stem cells form self-aggregating spheroids;
differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium;
differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells in a second medium.
2. The method of claim 1, wherein differentiating said pluripotent cells into hematopoietic endothelial cells is performed on a substrate under adherent conditions.
3. The method of claim 2, wherein the substrate comprises laminin.
4. The method of claim 2, wherein the substrate is attached to a two-dimensional surface.
5. The method of claim 2, wherein the substrate is attached to a three-dimensional structure.
6. The method of claim 1, wherein differentiating the pluripotent cells into hematopoietic endothelial cells is performed under low-adhesion or non-adhesion conditions to enable the hematopoietic endothelial cells to self-aggregate.
7. The method of claim 1, wherein the first medium comprises one or more of bone morphogenic protein 4(BMP4), basic fibroblast growth factor (bFGF), and Vascular Endothelial Growth Factor (VEGF).
8. The method of claim 7, wherein the first medium further comprises a WNT modulator.
9. The method of claim 1, wherein the second medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin.
10. The method of claim 1, wherein the pluripotent stem cells are human-induced pluripotent stem cells.
11. The method of claim 1, further comprising the step of harvesting and dissociating the expanded pluripotent stem cells.
12. The method of claim 1, further comprising the step of seeding the megakaryocyte progenitor cells onto a non-adherent surface in a culture medium prior to differentiating the megakaryocyte progenitor cells into megakaryocytes.
13. The method of any one of claims 1-12, further comprising the step of differentiating the megakaryocyte progenitor cells into megakaryocytes in a third medium.
14. The method of claim 13, wherein the third medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), interleukin-6 (IL-6), interleukin 9(IL-9), and heparin.
15. A method for producing megakaryocytes, comprising:
differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium; and
differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells in a second medium,
wherein at least one of said differentiated pluripotent cells and said differentiated hematopoietic endothelial cells are carried out on a three-dimensional structure coated with a matrix.
16. The method of claim 15, wherein the three-dimensional structure is a microcarrier.
17. The method of claim 15, wherein the three-dimensional structure is a microcarrier.
18. The method of claim 15, wherein the substrate comprises laminin.
19. The method of claim 15, wherein the first medium comprises one or more of bone morphogenic protein 4(BMP4), basic fibroblast growth factor (bFGF), and Vascular Endothelial Growth Factor (VEGF).
20. The method of claim 15, wherein the first medium further comprises a WNT modulator.
21. The method of claim 15, wherein the second medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin.
22. The method of claim 15, wherein the pluripotent stem cells are human-induced pluripotent stem cells.
23. The method of claim 15, further comprising expanding pluripotent stem cells on the matrix-coated three-dimensional structure.
24. The method of any one of claims 15-23, further comprising the step of differentiating the megakaryocyte progenitor cells into megakaryocytes in a third medium.
25. The method of claim 24, wherein the third medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), interleukin-6 (IL-6), interleukin 9(IL-9), and heparin.
26. A method for producing megakaryocytes, comprising:
differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium; and
differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells in a second medium,
wherein at least one of the differentiated pluripotent cells and the differentiated hematopoietic endothelial cells is performed under low-adhesion or non-adhesion conditions to enable the cells to self-aggregate.
27. The method of claim 26, wherein the first medium comprises one or more of bone morphogenic protein 4(BMP4), basic fibroblast growth factor (bFGF), and Vascular Endothelial Growth Factor (VEGF).
28. The method of claim 27, wherein the first medium further comprises a WNT modulator.
29. The method of claim 26, wherein the second culture medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin.
30. The method of claim 26, wherein the pluripotent stem cells are human-induced pluripotent stem cells.
31. The method of any one of claims 26-30, further comprising the step of differentiating the megakaryocyte progenitor cells into megakaryocytes in a third medium.
32. The method of claim 31, wherein the third medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), interleukin-6 (IL-6), interleukin 9(IL-9), and heparin.
33. A composition comprising a megakaryocyte progenitor cell produced by the method of any one of claims 1-12, 15-23, or 26-30.
34. A composition comprising a lysate of megakaryocyte progenitor cells produced by the method of any one of claims 1-12, 15-23, or 26-30.
35. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of any one of claims 13, 14, 24, 25, 31, or 32.
36. The composition of claim 35, wherein said megakaryocytes are CD42b+、CD61+And DNA+。
37. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of claim 13.
38. The composition of claim 37, wherein the megakaryocyte is CD42b+、CD61+And DNA+。
39. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of claim 14.
40. The composition of claim 39, wherein the megakaryocyte is CD42b+、CD61+And DNA+。
41. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of claim 24.
42. The composition of claim 41, wherein said megakaryocyte is CD42b+、CD61+And DNA+。
43. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of claim 25.
44. The composition of claim 43, wherein said megakaryocytes are CD42b+、CD61+And DNA+。
45. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of claim 31.
46. The composition of claim 45, wherein said megakaryocytes are CD42b+、CD61+And DNA+。
47. A composition comprising a megakaryocyte or a megakaryocyte lysate produced by the method of claim 32.
48. The composition of claim 47, wherein said megakaryocytes are CD42b+、CD61+And DNA+。
Technical Field
The present disclosure relates to methods of producing megakaryocyte progenitor cells and megakaryocytes, compositions having megakaryocyte progenitor cells and megakaryocytes, and uses thereof.
Background
Platelets are blood cells responsible for clot formation and vascular repair at sites of active bleeding. Physiologically, platelets are produced in the bone marrow from parent cells called Megakaryocytes (MK), which account for < 0.1% of the cells in the bone marrow. Mature MK is located outside sinusoidal blood vessels (sinuous blood vessels) in the bone marrow and extends a long structure called a proplatelet (proplatelet) into the circulatory system. The pre-platelets act as an assembly line for platelet production and release platelets sequentially from their ends.
MK is produced in the bone marrow by hematopoietic stem cells through a multi-step differentiation process. Exposure to various cytokines, chemokines, and growth factors, including thrombopoietin, results in differentiation of hematopoietic stem cells into pluripotent progenitor cells, followed by differentiation into committed megakaryocyte progenitor cells, also known as pre-MKs. Mature MK are produced by further differentiation, including cell expansion, increased DNA content, endomitosis, and granule formation. MK converts its cell mass into pre-platelet extension (extension) to produce/release anucleated platelets.
Low platelet counts are a significant consequence of a variety of diseases and therapies, including cancer treatment, transplantation and surgery, for which platelets are a critical first-line therapy to prevent death due to uncontrolled bleeding. Platelet units (3X10^11 platelets/200-. However, at this temperature, there is a risk of bacterial growth, which limits the shelf life of platelet units to 5 days, of which 2 days are spent by pathogen screening and 1 day spent in transport. Thus, blood centers typically do not have a platelet inventory available for transfusion for more than 1.5 days, which is rapidly depleted in emergency situations. A shortage of platelet donors may occur, especially at critical times. The increasing demand for civilian use alone exceeds the supply by about 20% and the stock will quickly run out in an emergency. In addition, extensive functional differences between units and donors lead to excessive transfusions to ensure effective bleeding control. Therefore, there is an urgent need to provide platelet-derived megakaryocytes, and novel and improved methods for producing megakaryocytes.
Disclosure of Invention
The present disclosure provides methods for producing megakaryocyte progenitor cells (pre-MKs) and Megakaryocytes (MKs) from stem cells. The disclosure further provides compositions comprising pre-MK and lysates thereof, and methods of using pre-MK, lysates thereof, and compositions thereof.
In some embodiments, the present disclosure provides methods for producing megakaryocytes, comprising: expanding pluripotent stem cells under low-adhesion or non-adhesion conditions and with agitation, wherein the expanded pluripotent stem cells form self-aggregating spheroids; differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium; differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells in a second medium. Differentiation of pluripotent cells into hematopoietic endothelial cells is performed on a substrate under adherent conditions. In some embodiments, differentiating the pluripotent cells into hematopoietic endothelial cells is performed under low-adhesion or non-adhesion conditions to enable the hematopoietic endothelial cells to self-aggregate.
In some embodiments, the present disclosure provides methods for producing megakaryocytes, comprising: differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium; and differentiating the hematopoietic endothelial cells into megakaryocytic progenitor cells in the second culture medium, wherein at least one of differentiating the pluripotent cells and differentiating the hematopoietic endothelial cells is performed on the matrix-coated three-dimensional structure. The three-dimensional structure may be a microcarrier or a microcarrier.
In some embodiments, the present disclosure provides methods for producing megakaryocytes, comprising: differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium; and differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells in the second medium, wherein at least one of differentiating the pluripotent cells and differentiating the hematopoietic endothelial cells is performed under low-adhesion or non-adhesion conditions to enable the hematopoietic endothelial cells to self-aggregate.
In some embodiments, the first medium comprises one or more of bone morphogenic protein 4(BMP4), basic fibroblast growth factor (bFGF), and Vascular Endothelial Growth Factor (VEGF). The first medium may include a WNT modulator. In some embodiments, the second medium comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), Fms-
In some embodiments, the pluripotent stem cell is a human-induced pluripotent stem cell.
In some embodiments, the method further comprises the step of expanding the pluripotent stem cells on the matrix-coated three-dimensional structure.
In some embodiments, the method may further comprise the step of differentiating the megakaryocyte progenitor cells into megakaryocytes in a third medium. The third medium may comprise one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-9 (IL-9), and heparin.
In some embodiments, the disclosure provides compositions having megakaryocyte progenitor cells or lysates of megakaryocyte progenitor cells produced by the methods of the disclosure.
In some embodiments, the disclosure provides megakaryocytes or megakaryocyte lysates produced by the methods of the disclosure. In some embodiments, such megakaryocytes are CD42b+、CD61+And DNA+。
Other features and advantages of the disclosure will be apparent from the following description, and from the claims.
Brief description of the drawings
The present disclosure will be described in detail by way of non-limiting examples of exemplary embodiments with reference to the accompanying drawings (wherein like reference numerals represent like parts of the several views of the drawings), and in which:
FIG. 1 shows a general schematic for large-scale differentiation of megakaryocyte progenitor cells (pre-MK), Megakaryocytes (MK), and Platelets (PLT) from the iPSC line.
Figure 2 depicts an example of a protocol for directed differentiation of pluripotent stem cells into multinucleated cells in a 2D matrix-dependent system such as a cell culture plate or flask.
Fig. 3A and 3B depict the pluripotency of 3 exemplary clinical grade hiPSC lines (referred to herein as PBG1, PBG2, PBG 3). Fig. 3A depicts low magnification phase contrast (phase contrast) images of PBG1, PBG2, and PBG3 ipscs forming characteristic growth regions when cultured on Vitronectin (Vitronectin) matrix with Essential8 medium. Fig. 3B depicts a larger magnification image of PBG1, PBG2 and PBG3iPSC immunostained for pluripotency factors Oct4 and Nanog and counterstained with a nuclear dye.
Fig. 4A, fig. 4B, fig. 4C and fig. 4D depict the culture of PBG1, PBG2 and PBG3iPSC, which undergo a stage of directed differentiation from pluripotent stem cells to mature megakaryocytes. FIG. 4A depicts a general timeline diagram of the directed differentiation process. Fig. 4B shows actual images of PBG1 cultures during phase 0 (day 0), phase I (
Fig. 5 is a graph depicting the average CD31+ differentiation efficiency of committed differentiation of PBG1, PBG2, and PBG3iPSC lines at the end of stage I.
Fig. 6A, 6B and 6C show the purity and yield of megakaryocyte progenitor cells (CD43+ CD41+) produced during stage II of committed differentiation of PBG1, PBG2 and PBG3iPSC lineage. FIG. 6A depicts representative CD41/CD43 flow cytometry data (from left to right: PBG1, PBG2, PBG3) for suspension cells harvested from stage II culture on
FIGS. 7A, 7B and 7C depict the final stage, stage III, of the committed differentiation protocol that ultimately produces mature megakaryocytes. FIG. 7A depicts the maturation status of PBG1, PBG2, and PBG 3-derived cells over time in stage III culture, as measured by the proportion of CD41+ megakaryocyte lineage cells that also express the mature megakaryocyte marker CD42 b. Fig. 6B and 6C depict light microscope images of hiPSC-derived megakaryocytes differentiated from PBG1 and PBG2, respectively, at stage III
Fig. 8A, 8B, 8C, 8D and 8E depict the characterization of mature megakaryocytes derived by differentiation of PBG1, PBG2 and PBG3 hipscs. FIG. 8A depicts stage III cells derived from PBG1 immunostained for
Fig. 9A, 9B and 9C depict the expansion of pluripotent PBG1 cells on recombinant vitronectin using various growth media. Figure 9A shows PBG1 growth in Essential8 medium. Figure 9B shows PBG1 growth in StemFlex medium. Figure 9C shows Nutristem XF medium PBG1 growth.
Fig. 10A, 10B and 10C depict flow cytometry data evaluating expression of the pluripotency markers Tra-1-60, SSEA5 and differentiation marker SSEA1 on PBG1 cells expanded using recombinant vitronectin on various recombinant media. Figure 10A shows pluripotency marker data from PBG1 cells expanded in Essential8 medium. Figure 10B shows pluripotency marker data from PBG1 cells expanded in StemFlex medium. Figure 10A shows pluripotency marker data from PBG1 cells expanded in Nutristem XF medium.
Fig. 11A, 11B and 11C depict the amplification of PBG1 ipscs in self-aggregating spheroid culture in a 3D stirred tank (without matrix). FIG. 11A shows microscope images of PBG1 spheroids in culture as a function of time. FIG. 11B depicts the increase in cell density over time in PBG1 spheroid culture. FIG. 11C depicts the mean PBG1 spheroid size over time in 3D culture.
FIGS. 12A, 12B depict flow cytometry data assessing expression of the pluripotency markers Tra-1-60, SSEA5 and the differentiation marker SSEA1 on PBG1 cells expanded in self-aggregating spheroid culture in a 3D stirred tank (no matrix). Figure 12A shows pluripotency marker data for PBG1 cells after a single 7 day expansion in a 3D stirred tank. FIG. 12B shows pluripotency marker data for PBG1 cells after 6-7 consecutive days of expansion in a 3D stirred tank.
Fig. 13A and 13B depict PBG1 ipscs immunostained against pluripotency factors Oct4 and Nanog and counterstained with nuclear dye. Figure 13A depicts part of a 2D colony of PBG-1 ipscs grown on vitronectin. Figure 13B depicts spheroids of PBG-1 ipscs grown under 3D stirring conditions (without matrix).
FIG. 14 depicts karyotyping of metaphase chromosome expansion of PBG 1iPSC spread (spread) grown by 4 consecutive 6-7 days of amplification from a 3D stirred tank, demonstrating normal karyotype after 4 rounds of 3D passaging.
Figure 15 depicts the morphological changes that occurred within 6 days of stage I differentiation of PBG1 ipscs to hematopoietic endothelial cells on collagen IV matrix in 2D culture vessels.
Fig. 16A and 16B depict representative stage I differentiation data for PBG 1-derived cells. Figure 16A depicts a representative flow cytometry analysis of PBG 1-derived cells at
Fig. 17A, 17B and 17C depict representative phase II data from PBG1 differentiation cultures. FIG. 17A shows phase II culture at
FIGS. 18A and 18B depict the average composition characteristics of stage II suspension cells. FIG. 18A depicts the average daily purity of pre-MK released over 10 days of phase II (i.e., CD41+ CD43+ CD 14-viable suspension cells%). Figure 18B depicts the median, quartile and range of contaminating myeloid progenitor cells (i.e., CD43+ CD14+ viable suspension cells%) within 10 days of phase II. All cultures were started with PBG1 cells on collagen IV matrix in 2D containers. Data represent 41 independent differentiations.
FIGS. 19A and 19B depict the production of released pre-MK. FIG. 19A depicts6 well equivalents each (i.e., 2ml medium, 9.5 cm) during phase II directed differentiation culture initiated using PBG 1iPSC are described2Surface area) of the pre-MK released (i.e., viability CD41+ CD43+ CD 14-). FIG. 19B depicts 6 well equivalents (i.e., 2ml of medium, 9.5 cm) between 6+4 and 6+8 of phase II committed differentiation culture initiated using PBG 1iPSC2Surface area) of the released pre-MK (i.e., viability CD41+ CD43+ CD 14-). Each dot represents an independent directed differentiation culture of PBG1 on collagen IV matrix in a 2D culture vessel.
Fig. 20A, 20B, 20C, and 20D depict MK differentiation and pre-platelet production in phase II. FIG. 20A depicts stage III,
Fig. 21A, 21B and 21C depict representative flow cytometry analyses from stage III
Fig. 22A and 22B show the use of laminin 521 and collagen IV in stage I of committed differentiation of PBG1 cells. FIG. 22A shows a signal at 4.2ug/cm2The process of stage I differentiation on human collagen IV. FIG. 22B shows the signal level at 0.13ug/cm2The process of stage I differentiation on recombinant human laminin 521.
FIGS. 23A, 23B and 23C depictThe use of recombinant laminin 521 to support megakaryocyte progenitor cell production and release during stage II of committed differentiation of PBG1 cells is described. FIG. 23A depicts the results from using 4.2ug/cm2Representative flow cytometry data for phase II of PBG1 differentiation culture of supportive matrix of human collagen IV. FIG. 23B depicts the results from using 0.13ug/cm2Representative flow cytometry data for stage II of PBG1 differentiation culture of supportive matrix of recombinant human laminin 521. FIG. 23C depicts 6 well equivalents (i.e., 2ml of medium, 9.5 cm) between 6+4 and 6+8 of phase II committed differentiation culture2Surface area) released pre-MK (i.e., viability CD41+ CD43+ CD14-), initiated using PBG 1iPSC, using 4.2ug/cm2Human collagen IV or 0.13ug/cm2Human laminin 521.
FIGS. 24A and 24B show pre-platelet production from MK differentiated from pre-MK produced in culture from laminin 521. Phase III (
FIGS. 25A and 25B show flow cytometry subgroup breakdown for phase III (
Fig. 26A, 26B, and 26C depict immunofluorescence microscopy images of
Fig. 27A and 27B depict immunofluorescence microscopy images of phase II cultures on
Figure 28 is a schematic depicting a protocol for directed differentiation of PBG1 into megakaryocyte progenitors using a packed bed (packed bed) bioreactor strategy, 3D, matrix-dependent approach. In the embodiment described herein, a laminin 521-coated Raschig ring made of PTFE is used as a large carrier (macrocarrier) to constitute a packed bed.
FIG. 29 depicts stage I differentiation of PBG-1 iPSCs on laminin 521 coated Rachig loops on
FIG. 30 depicts stage II differentiation of PBG-1 iPSCs on laminin 521 coated Rachig loops on
Fig. 31A, 31B and 31C depict flow cytometry data from PBG1 differentiation stages performed efficiently on a Rachig loop substrate. Fig. 31A depicts phase I on
Fig. 32 is a schematic of an exemplary 3D, matrix independent directed differentiation method using self-aggregating iPSC-derived spheroids in a stirred tank.
Fig. 33A and 33B depict
Fig. 34A, 34B, 34C and 34D depict phase II in committed differentiation initiated with self-aggregating spheroids of PBG1 ipscs. FIG. 34A depicts self-aggregating PBG 1-derived spheroids at
Fig. 35A, 35B and 35C depict stage III culture differentiation from 3D matrix-dependent culture initiated by self-focusing spheroids of PBG1 ipscs. FIG. 35A depicts a representative flow cytometry analysis from stage III
FIG. 36 depicts the pre-platelet spreading of mature MK harvested from 3D self-aggregating spheroid differentiation culture. An example of anterior platelet stretching is indicated by arrows.
Figure 37 depicts PBG 1-derived megakaryocytes immunostained for the megakaryocyte-
Fig. 38A, fig. 38B, fig. 38C, fig. 38D, fig. 38E and fig. 38F depict PBG 1-derived megakaryocytes immunostaining for alpha-granule specific protein platelet Factor 4(PF4), Von Willebrand Factor (VWF), and for the megakaryocyte-specific cell surface marker CD61 and nucleus.
FIGS. 39A, 39B, 39C, 39D, 39E and 39F depict immunostaining of PBG 1-derived megakaryocytes against Dense Granule specific protein (Dense granular specific protein) LAMP1 and serotonin, and against the megakaryocyte-specific cell surface marker CD61 and nucleus.
Fig. 40A, 40B, 40C and 40D are electron micrographs showing PBG 1-derived megakaryocytes. Fig. 40A is an electron microscope image showing that PBG 1-derived megakaryocyte produces microparticles (see arrow example). FIG. 40B is an electron microscope image (arrows; inset magnification) showing PBG 1-derived megakaryocytes and multivesicular bodies. Fig. 40C is an electron microscope image showing PBG 1-derived megakaryocytes, which are characterized by a multilobal (multi-lobed) nucleus, glycogen particles, alpha-particles, and an invaginated membrane system. Fig. 40D is an electron microscope image showing endoplasmic reticulum and mitochondria of PBG 1-derived megakaryocytes.
Fig. 41A, 41B and 41C show characteristic gene expression changes occurring during the process of directional differentiation of PBG1 into megakaryocytes. For all expression analyses, expression in pluripotent PBG1 cells was set to 1 and all other expression values were expressed in corresponding terms. Fig. 41A shows relative gene expression of Oct4 (pluripotency-associated gene) in
Fig. 42A, 42B, and 42C provide size distributions of PBG 1-derived megakaryocytes. FIG. 42A depicts a representative example of
Fig. 43A and 43B provide ploidy measurements for PBG 1-derived megakaryocytes. Figure 43A depicts a representative example of DNA ploidy measurements performed on PBG 1-derived megakaryocytes. FIG. 43B compares DNA ploidy measurements of PBG1-MK with MK from other sources.
FIG. 44 provides a comparison of the presence or absence and concentration ranges of various factors in hipSC-MK lysates of megakaryocytes derived by the methods of the disclosure and certain controls.
Fig. 45A, 45B, and 45C depict hiPSC platelet production. Fig. 45A depicts flow cytometry analysis of anucleated and nucleated cells (top, left). Nucleated cells contain a large amount of CD41+CD42+Megakaryocytes (upper, right). Assessment of CD41 Using flow cytometry+、CD42+And anucleated cells positive for calcein AM, and platelets were gated by size (1-5 microns). Fig. 45B shows an example of evaluation of platelets harvested from megakaryocyte cultures by electron microscopy. FIG. 45C is a graph depicting well CD41 during stage III of the committed differentiation protocol described herein+CD42+Calcein AM+Graph of cumulative yield of platelet size particles.
While the above-identified drawing figures set forth embodiments of the disclosure, other embodiments are also contemplated, as noted in the discussion. The present disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.
Detailed Description
The present disclosure relates to compositions and methods for producing megakaryocyte progenitor cells (pre-MK) and Megakaryocytes (MK) from stem cells such as pluripotent stem cells (e.g., clinical grade human-induced pluripotent stem cells). The methods enable continuous production of pre-MK from hematopoietic endothelial cells for an extended time frame (up to 8 days or longer) which can then be differentiated into mature MK. pre-MK and MK derived by the methods can be distinguished by one or more of their size range, ploidy profile, biomarker expression, gene expression, particle composition and growth factor, cytokine and chemokine composition, or combinations thereof.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide the skilled artisan with a general definition of many of the terms used in this disclosure: singleton et al, Dictionary of Microbiology and molecular Biology (Dictionary of Microbiology and molecular Biology) (2 nd edition, 1994); cambridge scientific Technology dictionary (The Cambridge dictionary Science and Technology) (Walker, eds., 1988); the Association of genetic terms (The Glossary of genetics), 5 th edition, R.Rieger et al (eds.), Schprinegverger (Springer Verlag) (1991); and Hale and Marham, The Harle Collins Dictionary of biology (The Harper Collins Dictionary of biology), 1991. As used herein, the following terms have the meanings given below, unless otherwise indicated.
By "agent" is meant any small molecule compound, antibody, nucleic acid molecule or polypeptide or fragment thereof.
The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. The term "antibody fragment" refers to a portion of an intact antibody and refers to the epitope variable region of an intact antibody.
"alteration" or "change" refers to an increase or decrease. The change may be at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even as much as 70%, 75%, 80%, 90%, or 100%.
By "biological sample" is meant any tissue, cell, body fluid, or other material derived from an organism.
By "capture reagent" is meant an agent that specifically binds to a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
By "cellular composition" is meant any composition comprising one or more isolated cells.
"cell survival" means cell viability.
As used herein, "clinical grade" means a cell or cell line derived or obtained using current Good Manufacturing Practice (GMP) that allows clinical use in humans. GMP is a quality assurance system used in the pharmaceutical industry to ensure that the final product meets preset specifications. GMP covers the manufacture and testing of the final product. It requires traceability of raw materials and is produced following certified Standard Operating Procedures (SOP).
By "detectable level" is meant an amount of analyte sufficient to be detected using methods conventionally used to perform such assays.
By "detecting" is meant identifying the presence, absence or quantity of an object to be detected.
By "detectable label" is meant a composition that, when attached to a molecule of interest, enables its subsequent detection by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens.
By "disease" is meant any condition or disorder that impairs or interferes with the normal function of a cell, tissue or organ. Examples of diseases include any disease or injury that results in a reduction in cell number or biological function, including ischemic injury, such as stroke, myocardial infarction or any other ischemic event that causes tissue damage, peripheral vascular disease, wounds, burns, bone fractures, blunt trauma, arthritis and inflammatory diseases.
By "effective amount" is meant the amount of the agent required to produce the desired effect.
"fragment" means a portion of a polypeptide or nucleic acid molecule. This portion comprises (preferably) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
The terms "isolated," "purified," or "biologically pure" refer to a material that is free of components that normally accompany it in its natural state to varying degrees. "isolation" refers to the degree of separation from the original source or surrounding environment. "purified (purify)" means a degree of separation greater than separation. A "purified" or "biologically purified" protein is completely free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may refer to a nucleic acid or protein that produces substantially one band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins that can be purified separately.
By "isolated polynucleotide" is meant a nucleic acid (e.g., DNA) that does not contain genes that flank the genes in the naturally occurring genome of the organism from which the nucleic acid molecules of the disclosure are derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or as an independent molecule (e.g., a cDNA or genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding other polypeptide sequences.
By "isolated polypeptide" is meant a polypeptide of the present disclosure that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% by weight free of proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the formulation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present disclosure. Isolated polypeptides of the present disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such polypeptide; or by chemically synthesizing the protein. Purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
The term "hematopoietic endothelial cell" as used herein means a cell that is capable of differentiating to produce a hematopoietic cell type or an endothelial cell type, and which may optionally be derived from a pluripotent stem cell. Hematopoietic endothelial cells typically adhere to extracellular matrix proteins and/or other hematopoietic endothelial cells, and in some embodiments, can be characterized by expression of markers CD31 and
By "marker" is meant any protein or other epitope that has an alteration in expression level or activity associated with a characteristic or condition.
The term "megakaryocyte" (MK) as used herein refers to a large (e.g., 10 μm diameter or more) polyploid hematopoietic cell with a propensity to produce pre-platelets and/or platelets. One morphological feature of mature MK is the development of a large multilobal (multi-lobad) nucleus. Mature MKs can stop proliferation, but continue to increase their DNA content through endomitosis with a corresponding increase in cell size.
The term "megakaryocyte progenitor" (pre-MK) as used herein refers to a mononuclear hematopoietic cell that is committed to the megakaryocytic lineage and is a precursor of mature megakaryocytes. Megakaryocyte progenitors are typically found in, but not limited to, bone marrow and other hematopoietic locations, but can also be generated from pluripotent stem cells, such as by further differentiating hematopoietic endothelial cells that are themselves derived from pluripotent stem cells.
The term "microparticle" refers to a very small (< 1 micron) phospholipid vesicle shed by megakaryocytes or other cells. The microparticles may contain genetic material, such as RNA, and express extracellular markers of their parent cells. Megakaryocyte and platelet-derived microparticles may play a role in a variety of pathways including hemostasis and inflammation.
The term "platelets" (PLTs) refers to cells that are anucleate with a diameter of 1-3 microns but do contain RNA. Platelets can express CD41, CD42b, and CD61 on their cell surface. Inside, it contains alpha and dense particles, which contain factors such as P-selectin and serotonin, respectively. Platelets also have an open tubule system, which refers to the pathway that transports extracellular material into the cell and releases the material from the particles to the extracellular environment. They play a role in the regulation of hemostasis, primarily by participating in blood clotting, but have also been shown to play a role in inflammation.
The term "pre-platelet" (preplate) refers to cells that are anucleate and do contain RNA, with a diameter of 3-10 microns. In addition, pre-platelets are similar in morphology and superstructure to platelets and constitute an intermediate cellular stage produced by megakaryocytes that divide by cytoskeletal rearrangement to form individual platelets.
The term "proplatelet (proplatelet)" refers to a cytoplasmic extension derived from or just released from megakaryocytes. The pre-platelets divide by cytoskeletal rearrangement to form individual pre-platelets and platelets.
The term "pluripotent stem cell" includes embryonic stem cells, embryonic derived stem cells and induced pluripotent stem cells as well as other stem cells that have the ability to form cells from all three germ layers of the body, regardless of the manner in which the pluripotent stem cell is derived. A pluripotent stem cell is functionally defined as a stem cell that may have one or more of the following characteristics: (a) capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; (b) cell types capable of differentiating into all three germ layers (e.g., can differentiate into ectodermal, mesodermal and endodermal cell types); or (c) expresses one or more embryonic stem cell markers (e.g., expresses Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.).
The term "induced pluripotent stem cell" (iPS cell or iPSC) refers to a pluripotent stem cell generated by reprogramming a somatic cell by expressing a combination of reprogramming factors. Ipscs can be generated using fetal, postnatal, neonatal, juvenile or adult somatic cells. Factors that may be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as
As used herein, the terms "prevent," "defense," "preventing," "prophylactic treatment," and the like refer to reducing the likelihood that a disorder or condition will develop in a subject that does not have the disorder or condition but is at risk of developing the disorder or condition or is suspected of developing the disorder or condition.
"decrease/decrease" refers to a negative change of at least 10%, 25%, 50%, 75%, or 100%.
"reduce/reduce cell death" refers to reducing/reducing the propensity or likelihood of cell death. Cell death may be apoptosis, necrosis or by any other means.
By "reduced level" is meant that the amount of analyte in the sample is less than the amount of analyte in the corresponding control sample.
"reference/reference" refers to standard or control conditions.
By "specifically binds" is meant an antibody or compound that recognizes and binds a polypeptide of the present disclosure, but which does not substantially recognize and bind other molecules in a sample (e.g., a biological sample) that naturally includes a polypeptide of the present disclosure.
The term "subject" or "patient: refers to an animal that is the subject of treatment/therapy, observation or experiment. By way of example only, subjects include, but are not limited to, mammals, including, but not limited to, humans or non-human mammals, such as non-human primates, murine, bovine, equine, canine, ovine, or feline animals.
As used herein, the terms "treat," "treating," and the like refer to reducing or alleviating a disorder or associated symptoms. It will be understood that, although not exclusive, treatment of a disorder or condition need not completely eliminate the disorder, condition, or symptoms associated therewith.
"comprises," "comprising," "contains," and "has" and the like can have the meaning attributed to them by U.S. patent law and can mean "comprising," "includes," and the like; "consisting essentially of … …" or "consisting essentially of … …" likewise has the meaning assigned thereto by the united states patent law and the term is open-ended, allowing the presence of more than the stated contents, as long as the basic or novel features of the stated contents are not changed by more than the presence of the stated contents, but excluding the implementation of the prior art.
As used herein, the term "or" is to be understood as being inclusive, unless otherwise indicated or apparent from the context. As used herein, the terms "a", "an" and "the" are to be construed as either singular or plural unless otherwise indicated herein or apparent from the context.
As used herein, unless otherwise indicated herein or clearly from the context, the term "about" should be understood to be within the range normally allowed in the art, e.g., with an average standard deviation of 2. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value. All numerical values provided herein are modified by the term about, unless expressly stated otherwise herein.
Reference to a list of groups in any definition of a variable herein includes the definition of that group as any single element or combination of listed groups. Reference to an embodiment of a variable or aspect described herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Megakaryocyte
In humans, megakaryocytes are derived from CD34+Hematopoietic stem cells, said CD34+Hematopoietic stem cells are found primarily in bone marrow, but also in the yolk sac, fetal liver and spleen during early development. During MK differentiation, MK precursors undergo an endomitosis, which makes MKs polyploid through multiple DNA replication cycles without cell division, producing multilobal nuclei (polylobulated nuclei) with up to 128n copies of DNA. As MKs increase in size, they also complement their transcriptional and proteomic profiles, acquire a number of highly specialized particles, including alpha particles and dense particles, and develop highly reentrant membrane systems, which are markers of MK maturation and development.
To produce platelets, MK migrates to the vicinity of blood vessels in the bone marrow, through which they extend long structures called pre-platelets into the circulation. The pre-platelets serve as an assembly line for platelet production and release a plurality of anucleated platelets sequentially from their ends.
Although platelets are primarily responsible for the formation of blood clots at sites of active bleeding, it is becoming increasingly apparent that they also play an important role in wound healing, angiogenesis and innate immunity. In the field of stem cell-based therapy, platelets are ideal early candidates (entrans) because they: 1) the shelf life of the stock is short, so the clinical demand is large; 2) is anucleate and can be safely irradiated to kill any contaminating nucleated cells, thereby reducing the risk of teratoma formation; 3) for most (> 90%) platelet transfusions, HLA or blood type matching is not required, which would facilitate large-scale production of allogeneic human pluripotent stem cells (hpscs) for off-the-shelf therapy; 4) short life and well characterized, which would simplify planned clinical trials; 5) The transplantation is easy; and 6) would greatly benefit from aseptic production, as current donor-based platelet infusions are inherently susceptible to bacterial and viral contamination.
The current platelet demand exceeds the supply by about 20%, and the unmet demand in 2022 is expected to double more as population growth and aging will require more platelet-based processes, new drugs to reduce platelet numbers, and expansion of existing uses of platelet transfusions to improve healing time. In the united states, the Blood market is effectively oligopolistic, less than half of the market being controlled by the American Red Cross (American Red Cross) and the American's Blood Centers (American's Blood Centers), respectively, while the hemelxcel corporation is the third largest Blood supplier.
There is a vast additional potential market for pre-MK and its growth factors. For example, pre-MK, MK and lysates thereof may be used in cell culture, tissue regeneration, wound healing, drug delivery, and for skin rejuvenation in the cosmetic industry. As megakaryocytes produced by the methods described herein contain a number of growth factors, they may be the source of a therapeutic composition and/or used for a number of therapeutic purposes. The present disclosure addresses these needs by creating an extensible business platform that conforms to the current good production specifications (cGMP) to make human iPSC-derived human megakaryocytes and their products.
In vitro, MK may be rationally derived from a variety of major stem cell sources, such as pluripotent stem cells, hematopoietic stem cells, or other stem cell types.
In some embodiments, MK may be derived from pluripotent stem cells, including, but not limited to, Embryonic Stem Cells (ESC) (e.g., human embryonic stem cells) and Induced Pluripotent Stem Cells (iPSC) (e.g., human induced pluripotent stem cells). ESCs are pluripotent stem cells derived from the inner cell mass of an early preimplantation embryo called the blastocyst. ipscs are a class of pluripotent stem cells that can be produced from adult cells by inducing the timed expression of specific transcription factors. ipscs can be indefinitely expanded and maintained in culture and engineered to produce MK.
In some embodiments, MK may be derived from hematopoietic stem cells, including but not limited toLimited to, CD34+Cord blood stem cells (UCB cells) (e.g., human CD 34)+Cord blood stem cells), CD34+Mobilizing peripheral blood cells (MPB) (e.g., CD34+Human mobilizing peripheral blood) or CD34+ bone marrow cells. UCB cells are pluripotent stem cells derived from blood that remains in the placenta and attached umbilical cord after delivery. MPB cells are pluripotent stem cells derived from volunteers who are mobilized into the bloodstream by administration of G-CSF or similar agents.
In some embodiments, MK may be derived from other stem cell types, including, but not limited to, Mesenchymal Stem Cells (MSCs), such as adipose-derived mesenchymal stem cells (admscs), or mesenchymal stem from other sources.
AdMSC is derived from white adipose tissue, which is derived from the mesoderm during embryonic development and is present in every mammalian species throughout the body. ASCs can be used in a variety of applications due to their wide availability and ability to differentiate into other tissue types of the mesoderm (including bone, cartilage, muscle and fat).
In the present disclosure, stem cell culture is maintained independently of animal serum and/or embryonic fibroblast feeder cells (which thereby increase the risk of human immunogenic reactions) that may be contaminated with xenogeneic pathogens. Thus, serum-free, feeder cells-free alternatives are used in the methods of the invention to avoid the introduction of animal products into pre-MKs and MKs derived according to the methods of the invention to ensure safe and animal product-free conditions and products.
Generation method
FIG. 1 shows a general schematic of large scale differentiation of megakaryocyte progenitors (pre-MK), Megakaryocytes (MK), and Platelets (PLT) from one or more pluripotent stem cells. It should be noted, however, that while the methods of the invention are described in connection with pluripotent stem cells, in various embodiments, the pluripotent stem cells may be replaced or supplemented with other types of stem cells.
Stage 0: preparation for expansion and differentiation of human-induced pluripotent stem cells
Matrix-dependent amplification culture
For matrix-dependent expansion culture, clinical grade Pluripotent Stem Cells (PSCs) can be expanded into colonies by feeder cells-free culture on a supporting matrix in pluripotent stem cell culture medium. The supportive matrix may be a two-dimensional surface or a three-dimensional structure. In some embodiments, the clinical grade human-induced pluripotent stem cells may be human-induced pluripotent stem cells (ipscs), such as PBG1, PBG2, or PBG3, although other types of pluripotent stem cells, such as embryonic stem cells or other stem cells, may be used.
In some embodiments, as non-limiting examples, the supportive matrix can be recombinant vitronectin, recombinant laminin, matrigel, or any combination of the foregoing. In some embodiments, the pluripotent stem cell culture medium can be, for example, but not limited to, Essential8 medium (thermo fisher), StemFlex medium (thermo fisher), NutriStem medium (Biological Industries), or other media known in the art to support maintenance and growth of pluripotent cells. In some embodiments, the cells may be cultured to achieve fusion. In some embodiments, the cells can be cultured to achieve 30% to 90% confluence. In some embodiments, the cells are cultured to achieve a confluence of up to 60%, up to 65%, up to 70%, up to 75%. For example, cells are cultured to achieve about 70% confluence. After a predetermined maximum percent confluence is reached, the cells are harvested. In some embodiments, cells may be harvested in bulk by dissociation using 0.1mM to 5m MEDTA or similar chelators or reagents. For example, cells can be harvested using about 0.5mM EDTA. In some embodiments, the cells may be harvested in single cells, e.g., by dissociation with proteolytic enzymes, collagen hydrolytic enzymes, or a combination thereof. For example, by reaction with, for example, recombinant trypsin such as TrypLETMOr AccutaseTMDissociation cells can be harvested as single cells. To maintain/expand PSCs, harvested cells can be resuspended in pluripotent stem cell culture medium.
Matrix independent 3D amplification culture
For matrix-independent 3D expansion cultures, clinical-grade PSCs can be expanded into self-aggregating spheroids. In some embodiments, this may be achieved by seeding single cells at a density of about 0.1 to about 1.5 million/ml. For example, in some embodiments, single cells may be seeded at 0.5 million/ml.
In pluripotent stem cell culture media, cells can be subjected to continuous movement by slow agitation or gentle shaking under low-adhesion or non-adhesion conditions. In some embodiments, feeder cells-free and serum-free media may be used. The pluripotent stem cell medium may be, for example, but not limited to, Essential8 medium (ThermoFisher), StemFlex medium (ThermoFisher), NutriStem medium (biotechnology industries), or other similar medium known in the art to support maintenance and growth of pluripotent cells. In some embodiments, the PSC spheroids are cultured for about 5-7 days until a total cell density of about 3 million to about 1 million cells/ml is achieved and/or a median spheroid size of about 150 to about 350 μm is achieved. In some embodiments, PSC spheroids are cultured until a total cell density of 5 million cells/ml is reached. In some embodiments, PSC spheroids are cultured until a median spheroid size of about 250 μm is achieved. The culturing step may last for 4, 5, 6, 7 or 8 days. Where applicable, PSCs can be harvested in single cell form, e.g., by dissociation with proteolytic enzymes, collagen hydrolyzing enzymes, or a combination thereof. For example, by using, but not limited to, trypsin, a recombinant trypsin such as TrypLETM，AccutaseTMOr similar reagents known in the art can be dissociated to harvest cells in single cells. In some embodiments, single cells are used to initiate another 3D expansion culture and/or directed differentiation culture.
Preparation for differentiation
In some embodiments, to prepare for differentiation, PSC aggregates can be generated by partially dissociating PSC colonies from a matrix-dependent 2D culture, by partially dissociating PSC spheroids from a matrix-independent 3D culture, or by self-aggregating single PSCs generated by any method known in the art. In some embodiments, before differentiation begins, it may beThese aggregates are resuspended and cultured in a pluripotent stem cell medium, such as, but not limited to, Essential8 medium (seegmfly), StemFlex medium (seegmfly) or NutriStem medium (bio-industrial). In some embodiments, the culture medium may comprise a ROCK inhibitor, such as, but not limited to, Y27632, H1152, or a combination thereof. In some embodiments, cells can be differentiated at 37 ℃ with 5% CO before they begin differentiation2、20％O2Culturing for 0-72 hr.
For matrix-dependent cultures, aggregates may be allowed to attach to the surface. In some embodiments, the attaching step may be allowed to proceed for about 24 hours, although any time between 1 hour and 24 hours or longer may be used. In some embodiments, the surface may be pre-coated with collagen \ laminin or any other extracellular matrix protein. In some embodiments, human collagen IV can be used to coat a surface. In some embodiments, the substrate-coated surface may be 2D (e.g., the bottom of a plastic dish or culture flask). In some embodiments, the substrate-coated surface may be 3D (e.g., smooth or reticulated spherical micro-vehicles, macro-vehicles, such as lauchig rings). The cells on the 3D matrix-coated surface can then be cultured with continuous or discontinuous motion. For example, cells can be cultured in spinner flasks, stirred tank bioreactors, vertical wheel bioreactors, packed bed bioreactors, or fluidized bed systems under ultra-low adhesion static conditions.
For matrix-independent cultures, aggregates can be subjected to continuous movement by slow stirring or gentle shaking in a low-adhesion vessel. After 0 to 72 hours, e.g., about 24 hours, the cells can transition to differentiation stage I.
Stage I production of hematopoietic endothelial cells
In stage I, the prepared PSC may be differentiated into hematopoietic endothelial cells. Briefly, the pluripotent stem cell medium was removed and replaced with stage I differentiation medium. In some embodiments, the stage I differentiation medium may be animal-freeFractionated culture medium (ACF) comprising StemBanTMACF (Stem cell technologies, Cat. 09855) as basal medium supplemented with one or more growth factors including, for example, bone morphogenetic protein 4(BMP4), basic fibroblast growth factor (bFGF) and Vascular Endothelial Growth Factor (VEGF). In some embodiments, the basal medium is supplemented with 1-200ng/ml of one or more of BMP4 (e.g., 50ng/ml), bFGF (e.g., 50ng/ml), and VEGF (e.g., 50 ng/ml). The cells may be incubated under hypoxic conditions for 2-6 days (e.g., 37 ℃, 5% CO)2，5％O2) Then incubated under normal oxygen for 2-6 days (37 ℃, 5% CO)2，20％O2). In some embodiments, WNT modulators, e.g., WNT agonists or antagonists, may be added for the initial stages of differentiation. In some embodiments, a GSK3 inhibitor may be added for the initial stage of differentiation. In some embodiments, a GSK3 inhibitor or a WNT agonist, such as CHIR9998014, CHIR99021 or a combination thereof, may be added for the initial stage of differentiation, such as on days 1-2. In some embodiments, a WNT modulator may replace one or more growth factors for at least part of phase I. For example, in some embodiments, BMP4 may be added during the first 48 hours and may be dispensable during the remaining phase I when WNT modulators are present. In some embodiments, VEGF and bFGF may be dispensable for the first 48 hours when a WNT modulator is present. In some embodiments, a daily complete media exchange may be performed during the entire phase I by removing used media and replacing it with fresh phase I media. In some embodiments, a partial media exchange may be performed, with 10-95% of the used media removed and replaced with an equal volume of fresh stage I media. In some embodiments, additional volumes of fresh medium may be added, which has the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing fresh stage I media or adding fresh stage I media, specific media components are incorporated into the culture.
In2In D-dependent matrix culture, by
Generation of detached megakaryocyte progenitors (Pre-MK) from hematopoietic endothelial cells
In some embodiments, initiation of megakaryocyte progenitor (stage II) differentiation may occur 4 to 8 days after stage I. Briefly, stage I medium is removed and replaced with an equal volume of stage II medium, e.g., STEMdiffTMAPELTM2 basal medium (Stem cell technologies, Cat. No. 05275). Such stage II media may be supplemented with 1 and 200ng/ml of any one or more of the following: stem Cell Factor (SCF) (e.g., 25ng/ml), Thrombopoietin (TPO) (e.g., 25ng/ml), Fms-related
Cells were then incubated at 37 ℃ with 5% CO2、20％O2Incubation is carried out for at least 7 days and up to 12 or more days. For example, the cells may be incubated for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 days. In some embodiments, a daily partial media exchange may be performed, with 10-95% of the used media removed and replaced with an equal volume of fresh phase II media. In some embodiments, additional volumes of fresh medium may be added, which has the net effect of increasing the total volume of the culture. In some embodiments, specific media components may be incorporated into the culture instead of or in addition to fresh stage I media.
Within 2-3 days after the start of phase II, small, round, refractive cells appeared within the adherent hematopoietic endothelial cells and were eventually released into the supernatant (fig. 17A). These released cells may contain pre-MK, as defined by CD43 and CD41 expression on the cell surface, and lack CD14 expression. In 2D culture, floating and weakly attached phase II cells that appear on top of the adherent cell layer can be harvested daily by gently washing and collecting the medium into conical tubes. The half-medium change can be initiated by adding half the original volume of fresh phase II medium on top of the washed adherent cell layer. Aliquots of the cells collected in the culture medium can be removed for live cell counting and biomarker analysis by flow cytometry. The remaining cells can then be centrifuged at 200Xg for 5 minutes. After centrifugation, media exchange can be accomplished by adding half of the original volume of supernatant back to the adherent cell layer. In matrix-independent 3D culture, the released cells can be harvested by suspending agitation to allow spheroids to settle to the bottom of the vessel, and then collecting up to 90% of the culture medium and suspended cells into a tube (e.g., a conical tube) for centrifugation. Half of the original volume of fresh medium may then be added to the vessel and replenished with conditioned medium from the centrifuged cell supernatant. In some embodiments, additional volumes of fresh medium may be added, which has the net effect of increasing the total volume of the culture. In some embodiments, specific media components can be incorporated into the culture in place of fresh stage II media. The remaining supernatant was discarded and the cell pellet containing pre-MK was stored at-180 ℃ in Cryostor10 cryopreservation medium, or passed directly to stage III. Megakaryocyte progenitor cells collected at stage II are small, round, refractive cells that express CD43 and CD41 and lack expression of
Generation of mature Megakaryocytes (MK) from megakaryocyte progenitors
In some embodiments, differentiation of mature megakaryocytes can be initiated using PSC-derived pre-MKs generated as described above. Fresh or thawed megakaryocyte progenitor cells can be seeded onto non-adherent surfaces in stage III media, including, for example, StemBanTM-ACF. Non-adherent surfaces refer to surfaces to which most cells are not intended to adhere or attach, but which are mostly maintained in suspension. For example, such surfaces may be made of "ultra-low adhesion plastic" or may not be coated with extracellular matrix proteins to prevent or minimize adhesion of cells to the surface. In some embodiments, the stage III medium may be supplemented with 0 to 200ng/ml of each of one or more of the following: TPO (e.g., 25ng/ml), SCF (e.g., 25ng/ml), IL-6 (e.g., 10ng/ml), IL-9 (e.g., 10ng/ml) and heparin (e.g., 5 units/ml). In some embodiments, the stage III medium can be further supplemented with UM171, UM729, SR-1, SU6656, or any combination thereof.
The cells can then be incubated at 37 ℃ -40 ℃ (e.g., 39 ℃), 5% -20% CO2(e.g., 7% -10%) and 5% -20% O2The next 5 days. In some embodiments, a daily partial media exchange may be performed, with 10-95% of the used media removed and replaced with an equal volume of fresh stage III media. In thatIn some embodiments, the non-adherent surface is a 6-well ultra-low-adhesion plate. In some embodiments, the non-adherent surface is a gas-permeable membrane (e.g., G-
In some embodiments, in stage III, megakaryocyte progenitor cells can differentiate into mature MKs within days. In some embodiments, the size and ploidy of small, round, refractive cells that are initially homogeneous (fig. 21) begin to increase until 2-4 days (fig. 20). At the same time, MK producing pre-platelets can be easily observed (fig. 20). By day 3-4 of stage III, CD61 co-expressing the mature MK markers CD42a and CD42b+The proportion of (megakaryocytic lineage) cells increased dramatically and levels as high as 80-90% could be reached, depending on the starting hiSPC cell line (fig. 21). By day 4-5 of phase III, mature MK release platelets into the culture medium. These platelets can be collected, quantified and evaluated by flow cytometry and electron microscopy, confirming that they are indeed platelets (fig. 45).
3D system
Packed bed bioreactor (Packed bed bioreactor)
In some embodiments, the 3D expandable packed bed bioreactor may be used to produce one or more of pre-production MK, megakaryocytes, platelets, or megakaryocytes and platelets. In some embodiments, packed bed bioreactors may be used for stage I and stage II cultures. For example, a packed bed reactor can be used to differentiate PSCs into hematopoietic endothelial cells, and then produce pre-MKs. The packed bed reactor carrier may be micron-sized or large-sized (macro-sized) and may be formed of biocompatible plastics, metals, glass, or natural materials such as alginate. In some embodiments, the carrier is formed from PTFE in the shape of a Raschig ring, for example 1mm Raschig. In some embodiments, the carrier may be coated with a matrix as described above. In some embodiments, the carrier may be coated with a laminin, such as recombinant human protein laminin 521. In some embodiments, the pluripotent cells may be seeded onto the carrier in the form of a pellet. In some embodiments, phase I media may be removed or replaced by daily media exchange. In some embodiments, during stage I, the pluripotent cells may exhibit a growth zone inside the carrier in the packed bed reactor. In some embodiments, initial differentiation of pluripotent cells into hematopoietic endothelial cells (i.e., stage I of committed differentiation) and further differentiation and release of pre-MK (i.e., stage II of committed differentiation) may occur in the same vessel. For example, a packed bed bioreactor may comprise a large carrier coated with laminin 521 seeded with pluripotent cells such as PBG-1 ipscs. The packed bed may then be exposed to a continuous flow of culture medium to differentiate stage I into hematopoietic endothelial cells. After permeation through the packed bed, the media may be circulated through a conditioning chamber, where fresh media components may be added, and the oxygen/
At the completion of stage I, the media can be switched for stage II differentiation and pre-MK production and release. A suitably sized and shaped support, such as a 1mm Raschig ring, can achieve sufficient media flow and channel width to allow released cells to permeate through the packed bed and out of the reactor for collection and cryopreservation. In some embodiments, this design may reduce the shear forces experienced by the cells, since its perfusion-based design may allow for efficient media use and continuous collection of pre-MK upon release.
Self-aggregating spheroids in stirred tank bioreactors
In some embodiments, certain processing steps may be performed using scalable 3D protocols, which may include differentiation using self-aggregating spheroids suspended in stirred or shaken containers (fig. 32). In some embodiments, such containers may include a low-adhesion surface or a non-adhesion surface, i.e., a surface coated with a hydrophilic or neutral charge coating, to inhibit specific and non-specific cell immobilization on the surface, forcing the cells into suspension. Pluripotent cells can be dissociated into single cells and resuspended in pluripotent maintenance medium. In some embodiments, the maintenance medium may be supplemented with a Rock inhibitor, such as H1152 or other Rock inhibitor. The pluripotent cells can then be incubated in a low or non-adherent container and stirred under standard culture conditions (e.g., 37 ℃, 5% CO2, 20% O2). In some embodiments where agitation is provided, the incubation container may be placed on an orbital shaker, or a continuously agitated shake or spin flask, or a controlled stirred tank bioreactor may be used. Within 24 hours, pluripotent cells can self-aggregate to form spheroids of about 50-150um in diameter. As the agitation stops, the spheroids settle to the bottom of the vessel.
The medium may then be exchanged with stage I differentiation medium to promote differentiation into hematopoietic endothelial cells, and agitation may be resumed and incubated under hypoxic conditions (e.g., 37 ℃, 5% CO2, 5% O2). The media exchange may be performed periodically (e.g., daily) during which the spheroids may grow larger and develop specific structures and shapes. For example, as shown in fig. 33A, spheroids can be cultured for a total of 6 days (4 days at 37 ℃, 5% CO2, 5% O2, followed by 2 days at 37 ℃, 5% CO2, 20% O2). As shown in fig. 33A, on
To transition to stage II, agitation may be suspended and the spheroids may be allowed to settle to the bottom of the vessel. The medium may then be exchanged with a stage II differentiation medium to promote differentiation and release of the suspension cells. After which the suspension cells may be collected periodically (e.g., daily) and subjected to a partial media exchange. The medium may be collected and centrifuged. Approximately half the working volume of fresh phase II differentiation medium, as well as a sufficient volume of conditioned medium (i.e., the centrifuged supernatant) can be added to the spheroids to restore the original working volume. Cell pellets can be cryopreserved or transferred to stage III for maturation into mature MK.
Upon transition to static stage III culture, pre-MK from 3D self-aggregating spheroid culture can produce MK purities similar to those of 2D culture systems. Furthermore, stage III differentiated cultures generated from 3D self-aggregating spheroid cultures may contain cells that sharply increase in size and are capable of producing pre-platelets, consistent with their property of being truly megakaryocytes.
Transition to scalable system for phase III
In some embodiments, as described above, fresh or thawed pre-megakaryocyte MK may be seeded onto a low-adhesive or non-adhesive surface in stage III media. In some embodiments, such non-adhesive surfaces can be breathable films (e.g., G-). In some embodiments, the low adhesion or non-adhesion surface is a cell culture bag or container with gentle agitation. In either case, pre-MK (freshly harvested from stage II culture, thawed from cryopreserved stock solution) is suspended in stage III medium at a density of 50-1000 ten thousand per ml and introduced into the vessel. For example, the density of pre-MK may be 100-150 ten thousand per ml, 100-200 ten thousand per ml, 100-300 ten thousand per ml, 100-400 ten thousand per ml, 200-500 ten thousand per ml, 2-600 ten thousand per ml, 300-700 ten thousand per ml, 300-800 ten thousand per ml, 500-900 ten thousand per ml or 800-1000 ten thousand per ml. The cells are cultured for a total of 1-5 days (e.g., 3 days) to enable differentiation into mature MK. In some embodiments, a daily media exchange may be performed, with 10-95% of the used media removed and replaced with an equal volume of fresh stage III media. At the end of stage III culture, the resulting cells increase in size and ploidy and exhibit a number of characteristics indicative of mature megakaryocytes (e.g., as shown in fig. 34-42 and described below).
Megakaryocyte and product thereof
In some embodiments, the present disclosure provides megakaryocyte progenitor cells, megakaryocytes, pre-platelets, or platelets derived in vitro from a PSC cell or cell line. According to aspects of the present disclosure, megakaryocyte progenitor cells, megakaryocytes, pre-platelets, or platelets derived from a PSC cell or cell line are produced using the method of U.S. patent No. 9,763,984 or the bioreactor disclosed in international patent application No. PCT/US2018/021354, which are incorporated herein by reference in their entirety.
In some embodiments, the present disclosure provides an isolated population of cells comprising megakaryocytes or megakaryocyte progenitor cells.
In some embodiments, the present disclosure provides compositions comprising megakaryocytes or megakaryocytes. In some embodiments of the disclosure, compositions comprising megakaryocytes, megakaryocyte progenitor cells, or products thereof are disclosed.
According to some embodiments of the present disclosure, the megakaryocytes, megakaryocyte progenitor cells, or products thereof are homogeneous in shape, size, and/or phenotype. It is understood that the megakaryocytes, megakaryocyte progenitor cells, or products thereof of the present disclosure can include variability in biomarker expression, size, ploidy, quantity, and purity that is characteristically distinct from variability in the corresponding human cells. In some embodiments, such variability may be significantly lower. In some embodiments, the cell population can be made to have a desired variability, which can be lower or higher than that of native cells.
In some embodiments, the megakaryocyte progenitor cell (pre-MK) is characterized by expression of markers CD43 and CD41 and by the absence of CD14 (i.e., CD 14)+、CD41+、CD43+). Additional expression of CD42b might indicate that the megakaryocyte progenitor cells are eventually maturing towards mature megakaryocytes. In certain embodiments, the megakaryocyte progenitor cells produced in differentiation culture are non-adherent and free-floating in the culture medium.
In some embodiments, the megakaryocyte of the invention is CD42a+、CD42b+、CD41+、CD61+GPVI + and DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42a+、CD42b+、CD41+，CD61+And DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42b+、CD61+And DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42a+、CD61+And DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42a+、CD41+And DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42b+、CD41+、CD61+And DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42b+、CD42a+、 CD61+And DNA+One or more of (a). In some embodiments, the megakaryocyte of the invention is CD42b+、CD42a+、CD41+And DNA+One or more of (a). In some embodiments, the megakaryocyte is CD41+CD61+CD42b+GPVI+. In some embodiments, the megakaryocyte is CD41+CD61+CD42a+GPVI+。
In some embodiments, the megakaryocyte of the invention is CD61+And DNA+And a diameter of about 10-50 μm. In some embodiments, the megakaryocytes produced by the methods described herein have an average size of 10-20 μm, 11-19 μm, 12-18 μm, 13-17 μm, 14-16 μm, 14-15 μm. In some embodiments, the megakaryocytes produced by the methods described herein have an average size of 14.5 μm. In some embodiments, the megakaryocytes of the present invention are about 10-20 μm in diameter. In some embodiments, the megakaryocytes of the present invention are about 10-30 μm in diameter. In some embodiments, the megakaryocytes of the present invention are about 10-40 μm in diameter. In some embodiments, the megakaryocytes of the invention are about 10-50 μm in diameter. In some embodiments, the megakaryocytes of the invention are about 20-40 μm in diameter. In some embodiments, the megakaryocytes of the invention are about 25-40 μm in diameter.
In some embodiments, the megakaryocytes produced by the methods described herein have a ploidy of 2N to 16N. In some embodiments, the megakaryocytes of the invention have a ploidy of at least 4N, 8N, or 16N. In some embodiments, megakaryocytes of the invention have a ploidy of 4N-16N. In some embodiments, the megakaryocytes produced by the methods described herein are 16% +/-11.4% CD61+ cells at 72 hours in phase III culture, with more than 4N DNA.
In some embodiments, at least 50% of the megakaryocyte population produced by the methods described herein is CD61+And DNA+And has a ploidy of 2N-16N. For example, megakaryocytes (i.e., beta-1-tubulin positive stage III cells) from differentiated cultures representing PBG1 range in size from about
In some embodiments, the isolated cell population or composition comprises at least 50% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 55% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 65% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 60% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 70% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 75% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 80% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 85% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 90% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 95% CD42b+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 98% CD42b+CD61+DNA+A cell.
In some embodiments, the isolated cell population or composition comprises at least 50% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 55% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 65% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 60% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 70% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 75% CD42b+CD41+CD61+A cell. In some embodiments, the isolated cell population or composition comprises at least 80% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 85% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 90% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 95% CD42b+CD41+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 98% CD42b+CD41+CD61+DNA+A cell.
In some embodiments, the isolated cell population or composition comprises at least 50% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 55% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 65% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 60% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 70% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 75% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 80% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 85% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 90% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 95% CD42b+CD42a+CD61+DNA+A cell. In some embodiments, the isolated cell population or composition comprises at least 98% CD42b+CD41+CD61+DNA+A cell.
In some embodiments, the isolated population or composition of cells comprises at least 50% megakaryocytes having a ploidy of 4N or greater. In some embodiments, at least 50% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 60% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 70% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 80% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 90% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, the isolated population of cells or composition comprises megakaryocytes having an average ploidy of 4N.
In some embodiments, the isolated population of cells or composition comprises pre-platelets, or platelets produced by the megakaryocytes of the present disclosure. In some embodiments, the pre-platelets, or platelets are CD42b+CD61+DNA-A cell. In some embodiments, the megakaryocytes are produced in vitro by differentiating the hiPSC cells or cell lines.
In some embodiments, the megakaryocytes produced by the methods described herein comprise one or more of the following: (a) content of MK particles by immunofluorescence microscopy: PF4 and VFW for alpha-particles and LAMP-1 and serotonin for dense particles; (b) gene expression data: oct4-, Nanog-, Sox2-, Zfp42-, Zfpm1+, Nfe2+, Runx1+, Meis1+, Gata1 +; (c) has low/no fibrinogen, serotonin and LDL, and (d) can take up fibrinogen, serotonin and LDL when incubated with plasma.
In some embodiments, the megakaryocytes produced by the methods described herein have a characteristic expression profile of growth factors, cytokines, chemokines, and related factors (fig. 44). In some embodiments, the disclosure provides compositions or pharmaceutical compositions comprising megakaryocytes of the invention, which can include factors such as platelet-derived growth factor isoforms PDGF-AA or PDGF-BB, Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factor Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), CXC chemokine family member CXCL1(GRO α) or CXCL12(SDF-1), TNF superfamily member sCD40L or TRAIL or CC chemokine family member CCL5(RANTES), CCL11 (eotaxin-1), CCL21(6CKine) or CCL24 (eotaxin-2). In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising a megakaryocyte lysate of the present invention. Such lysates may be prepared by any method known in the art, such as disrupting the pre-MK or a membrane of MK, by a virus, enzyme, or osmotic mechanism that compromises its integrity. In some embodiments, the lysate may include other reagents or be prepared in different compositions (liquids, pastes, etc.) as desired for a particular application. In some embodiments, such compositions can comprise factors such as platelet derived growth factor isoforms PDGF-AA or PDGF-BB, Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factor Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), CXC chemokine family members CXCL1(GRO α) or CXCL12(SDF-1), TNF superfamily member sCD40L or TRAIL or CC chemokine family member CCL5(RANTES), CCL11 (eotaxin-1), CCL21(6CKine), or CCL24 (eotaxin-2).
Application method
In some embodiments, pre-MKs and components thereof of the present invention may be a source of growth factors, such as human growth factors. In some embodiments, such growth factors are useful in cell culture, tissue regeneration, wound healing, bone regeneration, cosmeceutics, and hemostatic bandages. In some embodiments, the megakaryocytes or lysates thereof or compositions thereof of the invention can be used in cell culture. In some embodiments, the megakaryocytes or lysates thereof or compositions thereof of the present invention can be used as cosmeceuticals. In some embodiments, the megakaryocytes or lysates thereof or compositions thereof of the invention can be used as therapeutic agents. For example, megakaryocytes or lysates thereof or compositions thereof of the invention can be used to increase ex vivo cell expansion, improve bone marrow regeneration in vivo, increase tissue regeneration and vascularization, and increase survival in animals in radiological studies.
In some embodiments, pre-MKs and MKs of the invention may be used to generate platelets to support current transfusion needs (e.g., surgery, chemotherapy, pregnancy/labor, trauma). National defense and safety measures are a high priority in the united states and, as a radiation countermeasure, represent a large potential market for MK and its products. Radiation exposure (such as may occur following a nuclear accident or challenge) inhibits platelet production. A large radiation event will trigger an immediate demand for platelets, which will deplete existing local inventory to treat emergency wounds, as well as a sustained demand for platelets for survivors more than 6 days after exposure. With the reduction of platelets in the affected population, national strategic platelet reserves will become very important as the gap in preparation for our military will shift from the front line to 24-48 hours after the event occurs. Platelet inventory is not maintained in the united states in the national Strategic stock repository (Strategic national stock) and there are no approved therapeutics that immediately increase platelet counts. pre-MKs and MKs according to some embodiments may be used for on-demand platelet production. The ability to stock the pre-MKs for long periods of time and develop the on-demand hiPSC-platelet production capacity would enable the establishment of strategic national stocks of hipscs-platelets, which is crucial to meet this anticipated demand.
It has been demonstrated that media supplemented with autologous Platelet Rich Plasma (PRP) can culture microvascular endothelial cells, thereby improving the vascular integrity of the perfused transplanted organ. Platelets store biologically active factors in secretory granules, which are obtained from megakaryocytes. Inclusion includes various chemokines and growth factors, such as platelet-derived growth factor isoforms (PDGF-AA, -AB and-BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), Hepatocyte Growth Factor (HGF), Connective Tissue Growth Factor (CTGF), and
Reagent kit
The present disclosure provides kits comprising megakaryocytes or differentiated cells of the present disclosure. In one embodiment, the kit comprises a composition comprising isolated megakaryocytes. In particular embodiments, the present disclosure provides kits for differentiating, culturing, and/or isolating megakaryocytes or precursors thereof of the present disclosure. In certain embodiments, the present disclosure provides kits for producing platelets.
In some embodiments, the kit comprises a sterile container containing the cell composition; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable containers known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other material suitable for containing a medicament.
If desired, the kit can be provided with instructions for producing megakaryocytes. The instructions generally include information regarding the conditions and factors required to differentiate, culture and/or isolate megakaryocytes or precursors thereof. In some embodiments, instructions for producing platelets are included. The instructions may be printed directly on the container (if present), or provided in or with the container as a label applied to the container, or as a separate sheet, booklet, card or folder.
The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are explained fully in the literature, for example, molecular cloning: a laboratory Manual ("Molecular Cloning: laboratory Manual"), second edition (Sambrook, 1989); "" oligonucleotide synthesis "(Oligonucleotides Synthesis)" (Gait, 1984); "" Animal Cell Culture "(Animal Cell Culture)" (Freshney, 1987); "Methods in Enzymology" (Methods in Enzymology), "Handbook of Experimental Immunology" (Weir, 1996); "" Gene transfer Vectors for Mammalian Cells "(GeneTransfer Vectors for Mammalian Cells)" (Miller and Calos, 1987); "New Molecular Biology laboratory Manual (Current Protocols in Molecular Biology)" (Ausubel, 1987); "" PCR: polymerase Chain Reaction (PCR: The Polymerase Chain Reaction), (Mullis, 1994); "(Current Protocols in Immunology)" (Coligan, 1991). These techniques are suitable for producing the polynucleotides and polypeptides of the present disclosure, and thus can be considered in making and practicing the present disclosure. The following sections will discuss techniques that are particularly useful for particular embodiments.
The following examples are provided to fully disclose and describe to those of ordinary skill in the art how to make and use the assays, screens, and therapeutic methods of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.
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