CN117280021A

CN117280021A - Human urine-derived induced somite anterior mesodermal progenitor cells and uses thereof

Info

Publication number: CN117280021A
Application number: CN202280008197.6A
Authority: CN
Inventors: 裴端卿; 秦月; 黄星南; 曹尚涛
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS; Westlake University
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS; Westlake University
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2023-12-22
Also published as: WO2023133726A1

Abstract

Provided herein are methods of reprogramming or inducing somite pre-mesodermal progenitor cells from urine cells, somite structures formed from somite pre-mesodermal progenitor cells, and uses thereof.

Description

Human urine-derived induced somite anterior mesodermal progenitor cells and uses thereof

Technical Field

The present application relates to human urine-derived urine cell-induced somite anterior mesodermal progenitor cells (uipsms), somite structures formed by UiPSM cells, methods of producing UiPSM cells and somite structures, and uses thereof.

Background

Terminally differentiated somatic cells can be successfully reprogrammed to pluripotent stem cells, widening the field of research for reprogramming adult cells to stem cells (Takahashi et al, 2007; takahashi and Yamanaka, 2006). In addition, induced pluripotent stem cells (ipscs) have the potential to differentiate towards the germ layer, but their use is limited by the risk of tumorigenicity due to iPSC induced differentiated cells, which cannot reach 100% efficiency. Secondly, for human cells, many kinds of cells such as fibroblasts, blood cells, amniotic cells, skin epithelial stem cells, proximal tubule (HK 2) cell lines, pericyte-derived cells, endothelial cells, exocrine pancreatic cells, etc. are the sources of initiation of reprogramming induction, and the acquisition of these cells causes a certain degree of damage to the subject.

In addition, urine cells obtained non-invasively can be reprogrammed to induce pluripotent stem cells (ipscs) and Neural Progenitor Cells (NPCs) (Wang et al, generation of integration-free neural progenitor cells from cells in human urine, nat Methods 10, 84-89, 2013; zhou et al, generation of human induced pluripotent stem cells from urine samples, nat-Protoc 7, 2080-2089, 2012), suggesting that urine cells have strong plasticity as a starting cell, can be used as a starting cell for inducing differentiation, and has certain advantages in clinical applications.

Vertebrate embryos exhibit highly conserved tissue space pattern features. The most convincing evidence for dual fate neuromesodermal progenitor (NMP) comes from a population of tail bud cells with self-renewal capacity that will migrate to the neural tube and predetermined somite mesodermal sites for development (Henrique et al 2015). Somite anterior mesodermal cells (PSM) are axial stem cells derived from caudal ectoderm (CLE) and form somites along the anterior-posterior axis, which determine the extension of the body axis during embryonic development (Henrique et al 2015; saito and Suzuki, 2020). Paraxial mesoderm (also known as somite mesoderm) cells can develop into skeletal, myocyte and dermal cell lineages and differentiate further into musculoskeletal systems, but it is difficult to obtain self-renewing PSM cells in human embryos due to ethical and technical limitations. Thus, the in vitro construction of self-renewing PSM progenitors is of great interest for the study of somatic genesis. However, it is difficult to obtain self-renewing PSM cells in human embryos due to ethical and technical limitations.

Although human embryonic stem cells (ES) can differentiate into PSM and aggregate in defined conditioned media to generate a three-dimensional "gastrulation" model (Beccari et al, 2018; moris et al, 2020), the occurrence of gastrulation does not mimic the process of somite production well and is therefore not a good model for studying related diseases. This result is mainly due to the lack of PSM properties of actively aggregated cells, such as self-renewal and multipotent differentiation of somite mesodermal cells. Therefore, it is important to generate self-renewing PSM cells to construct somite structures to mimic somite genesis in early embryonic development.

Summary of The Invention

The present disclosure shows that urine cells can be reprogrammed to somite pre-mesodermal progenitor cells that can stably expand and differentiate into cells of the mesodermal lineage. The somite anterior mesodermal progenitor cells can also self-assemble to form somite-like structures to mimic somite genesis.

The resulting extraganglionic mesodermal progenitor cells are designated herein as urine-derived urine cell-induced extraganglionic mesodermal progenitor cells (abbreviated uippm). UiPSM cells highly express genes related to the anterior-mesoderm of somites, and have a characteristic expression profile of the anterior-mesoderm transcription of somites. Importantly, uiPSM cells exited the pluripotent state, reducing the risk of tumor formation, while retaining the potential to differentiate into cells of the somite anterior mesodermal lineage.

UiPSM cells can self-assemble in vitro to produce a somite (UiSomitoid) structure, a "coracoid" structure that is similar to a somite that UiPSM self-assembles. The UiSomitoid structure mimics the establishment of similar tissues in somite formation during early embryonic development, and is primarily involved in stem cell-based embryo model (SCME) research direction. The UiSomitoid structure better mimics the establishment of the anterior-posterior axis and molecular clocks of embryogenic body segments.

In one aspect, provided herein is a method of inducing urine cells to obtain extraganglionic mesodermal progenitor cells (UiPSM cells). Specifically, the method may comprise the steps of:

(a) Culturing urine cells and selecting epithelial-like cells from the cultured urine cells;

(b) Transforming an epithelial-like cell with a vector;

(c) Transformed epithelial-like cells are induced in basal medium supplemented with WNT agonist, DOT1L inhibitor and growth factor(s).

In some embodiments, the urine cells are obtained from an upper urinary tract urine sample collected from one or more subjects.

In some embodiments, urine cells are cultured with REGM medium and are enriched for mainly epithelial-like cells and mesenchymal cell types during the culture process.

In some embodiments, the epithelial-like cells are transformed by electroporation with one or more vectors to render the cells susceptible to subsequent induction. For example, the vectors used may be pEP4E02SET2K and pCEP4-miR-302-367. In some embodiments, the state of the urine cells is restored within 2 days after electroporation.

In some embodiments, the growth factor used is selected from FGF (e.g., bFGF), EGF, VEGF, PDGF, TGF-beta, PD-ECGF, TNF, HGF, IGF (e.g., IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.

In some embodiments, the basal medium used to induce the epithelial-like cells is Advanced DMEM/F12.

In some embodiments, the WNT agonist is CHIR99021.

In some embodiments, the DOT1L inhibitor is EPZ5676.

In some embodiments, induction is performed over a period of about 7 to 12 days.

In one aspect, provided herein are somite anterior mesodermal progenitor cells (UiPSM cells) obtained by the methods described herein. UiPSM cells have the ability to differentiate into cells of the extraganglionic mesoderm lineage, such as skeletal muscle cells, osteoblasts, chondrocytes and chondroblasts.

In one aspect, provided herein is a somite structure comprising a somite pre-mesodermal progenitor cell derived from a urine cell as described above. In particular, the somite structure is produced by in vitro expansion and differentiation of the somite pre-mesodermal progenitor cells. Preferably, the somite structure is a self-organizing "coracoid" structure.

In one aspect, provided herein is a method of producing a somite structure as described above, the method comprising:

(a) A quantity (e.g., 400) of UiPSM cells were seeded in wells of a low adhesion plate for aggregation during 48 hours;

(b) Extending the aggregated cells in a medium containing WNT agonist and NODAL inhibitor for up to 7 days;

(c) The "beak-tail" 3D somite structure was obtained from the plate.

In some embodiments, the WNT agonist is CHIR99021.

In some embodiments, the NODAL inhibitor is SB431542.

In one aspect, provided herein is the use of urine-derived somite pre-mesodermal progenitor cells (UiPSM cells) in inducing differentiation of skeletal muscle cells, osteoblasts and chondrocytes.

In one aspect, provided herein is a reprogramming system for inducing from urine cells into extraganglionic mesodermal progenitor cells (UiPSM cells).

In general, the present disclosure relates to the following embodiments:

1. a method of inducing urine cells to produce somite anterior mesodermal progenitor cells comprising the steps of:

(a) Culturing live urine cells for a suitable period of time to allow for the appearance of epithelial-like cells, and isolating said epithelial-like cells;

(b) Inducing the epithelioid cells in basal medium supplemented with WNT agonist, DOT1L inhibitor, and one or more growth factors for a time sufficient to form granulated colonies; and

(c) Extranodal mesodermal progenitor cells are obtained from the colonies.

2. The method of embodiment 1, wherein prior to step (b), the epithelial-like cells are pretreated with one or more vectors capable of improving the permeability of the cells for inducing or enhancing reprogramming of the cells.

3. The method of embodiment 2, wherein the vector expresses one or more of the following factors: oct4, sox2, SV40LT, klf4, miRNA 302, miRNA 303, miRNA 304, miRNA305, miRNA 306, and miRNA 307.

4. The method of embodiment 2 or 3, wherein after pretreatment, the epithelial-like cells are further cultured to restore their ability.

5. The method of any one of the preceding embodiments, wherein the urine cells are isolated from an upper urinary tract urine sample collected from one or more donor subjects.

6. The method of any one of the preceding embodiments, wherein in step (a), the urine cells are cultured in REGM medium.

7. The method of any one of the preceding embodiments, wherein in step (b), the basal medium is DMEM, DMEM/F12 or Advanced DMEM/F12.

8. The method of any one of the preceding embodiments, wherein the growth factor is selected from the group consisting of FGF (e.g., bFGF), EGF, VEGF, PDGF, TGF- β, PD-ECGF, TNF, HGF, IGF (e.g., IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.

9. The method of any one of the preceding embodiments, wherein the growth factor is bFGF and EGF.

10. The method of any one of the preceding embodiments, wherein the WNT agonist is CHIR99021 and/or the DOT1L inhibitor is EPZ5676.

11. The method according to embodiment 10, wherein the basal medium is Advanced DMEM/F12 supplemented with CHIR99021 at a concentration ranging from about 2-4. Mu.M (e.g., 3. Mu.M, 2.5-3. Mu.M, 3-3.5. Mu.M), EPZ5676 at a concentration ranging from about 4-6. Mu.M (e.g., 5. Mu.M, 4.5-5. Mu.M, 5-5.5. Mu.M), bFGF at a concentration ranging from about 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/. Mu.l) and EGF at a concentration ranging from about 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/. Mu.l) and 5-5.5 ng/. Mu.l.

12. The method of any of the preceding embodiments, wherein the induction in step (b) is performed for a period of about 7-12 days, e.g., about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, and about 12 days.

13. A somite anterior mesodermal progenitor cell obtained or obtainable by the method of any one of the preceding embodiments.

14. A method of culturing the somite pre-mesodermal progenitor cells of embodiment 13, comprising culturing the somite pre-mesodermal progenitor cells in a basal medium supplemented with a WNT agonist, a TGF- β inhibitor, and one or more growth factors.

15. The method of embodiment 14, wherein the basal medium is DMEM, DMEM/F12 or Advanced DMEM/F12.

16. The method of embodiment 14 or 15, wherein said WNT agonist is CHIR99021 and/or said TGF- β inhibitor is a8301.

17. The method of any of embodiments 14-16, wherein the growth factor is selected from the group consisting of FGF (e.g., bFGF), EGF, VEGF, PDGF, TGF- β, PD-ECGF, TNF, HGF, IGF (e.g., IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.

18. The method of embodiment 17, wherein the growth factors selected are bFGF and EGF.

19. The method according to embodiment 18, wherein the defined medium is Advanced DMEM/F12 supplemented with CHIR99021 in a concentration range of about 2-4. Mu.M (e.g., 3. Mu.M, 2.5-3. Mu.M, 3-3.5. Mu.M), A8301 in a concentration range of about 4-6. Mu.M (e.g., 5. Mu.M, 4.5-5. Mu.M, 5-5.5. Mu.M), bFGF in a concentration range of about 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/. Mu.l) and EGF in a concentration range of about 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/. Mu.l).

20. The method according to any one of embodiments 14-19, further comprising passaging the cultured somite pre-mesodermal progenitor cells.

21. A somite anterior mesodermal progenitor cell obtained or obtainable by the method of any of embodiments 14-20.

22. A method for differentiating somite anterior mesodermal progenitor cells into skeletal muscle cells, comprising:

(a) Inoculating the somite anterior mesodermal progenitor cells overnight in basal medium supplemented with WNT agonist, TGF- β inhibitor and one or more growth factors;

(b) Inducing cells in basal medium supplemented with KSR, ITS, NEAA, β -ME, IGF-1, HGF, CHIR99021, VC, dex and SB 431542; and

(c) Cells were induced in basal medium supplemented with KSR, horse serum, NEAA, beta-ME, IGF-1 and HGF.

23. The method of embodiment 22, wherein in step (b), the basal medium is supplemented with about 15% KSR, about 1% ITS, about 1% NEAA, about 0.1. Mu.M.beta. -ME, about 4ng/ml IGF-1, about 10ng/ml HGF, about 3. Mu.M CHIR99021, about 50ng/ml VC, about 0.5ng/ml Dex, and about 2nM SB431542.

24. The method according to embodiment 22 or 23, wherein in step (c) the basal medium is supplemented with about 15% KSR, about 2% horse serum, about 1% NEAA, about 0.1. Mu.M beta. -ME, about 4ng/ml IGF-1, and about 10ng/ml HGF.

25. The method of any one of embodiments 22-24, wherein in step (b), induction is performed for a period of 12-18 days, e.g., 15 days, 12 days, 13 days, 14 days, 16 days, 17 days, and 18 days.

26. The method of any one of embodiments 22-25, wherein in step (c), induction is performed until skeletal muscle fiber bundles are fully present.

27. A method for differentiating somite pre-mesodermal progenitor cells into osteoblasts, comprising:

(a) Inoculating the somite anterior mesodermal progenitor cells overnight in basal medium supplemented with WNT agonist, TGF- β inhibitor and one or more growth factors; and

(b) Cells were induced in basal medium supplemented with FBS, VC, beta-glycerophosphate and 1-thioglycerol.

28. The method of embodiment 27, wherein in step (b), the basal medium is supplemented with about 10% FBS, about 50ng/ml VC, about 100nM beta-glycerophosphate and about 1. Mu.M 1-thioglycerol.

29. A method for differentiating somite anterior mesodermal progenitor cells into chondrocytes, comprising:

(b) Cells were induced in basal medium supplemented with FBS, ITS, sodium pyruvate, VC, beta-glycerophosphate, TGF-beta 3 and BMP2.

30. The method of embodiment 29, wherein in step (b), the basal medium is supplemented with about 10% FBS, about 1% ITS, about 1% sodium pyruvate, about 50ng/ml VC, about 0.1nM beta-glycerophosphate, about 4ng/ml TGF-beta 3, and about 20ng/ml BMP2.

31. The method of any one of embodiments 27-30, wherein the induction is performed for about 15 days.

32. The method of any one of embodiments 22-31, wherein the basal medium is DMEM/F12 or DMEM.

33. The method of any one of embodiments 22-32, wherein the medium is exchanged every 2-3 days during induction.

34. A method for producing a somite structure, comprising:

(a) Seeding the somite pre-mesodermal progenitor cells of embodiments 13 or 21 and culturing for a time sufficient to form a compact spherical cell aggregate;

(b) Elongating cell aggregates in basal medium supplemented with WNT agonist and NODAL inhibitor; and

(c) The elongated structure is separated from the plate.

35. The method of embodiment 34, wherein in step (a), the culturing is performed in a medium supplemented with WNT agonist, TGF- β inhibitor, and one or more growth factors.

36. The method of embodiment 35, wherein the medium is Advanced DMEM/F12 supplemented with CHIR99021, a8301, bFGF and EGF.

37. The method of embodiment 36, wherein the medium is Advanced DMEM/F12 supplemented with CHIR99021 at a concentration ranging from about 2-4. Mu.M (e.g., 3. Mu.M, 2.5-3. Mu.M, 3-3.5. Mu.M), A8301 at a concentration ranging from about 4-6. Mu.M (e.g., 5. Mu.M, 4.5-5. Mu.M, 5-5.5. Mu.M), bFGF at a concentration ranging from about 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/. Mu.l) and EGF at a concentration ranging from 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/. Mu.l).

38. The method of embodiment 34, wherein in step (b), the basal medium is Advanced DMEM/F12 supplemented with CHIR99021 and SB 431542.

39. The method of embodiment 38, wherein the concentration of CHIR99021 ranges from about 2 to 4 μm (e.g., 3 μm, 2.5 to 3 μm, 3 to 3.5 μm) and the concentration of SB431542 ranges from about 5 to 15 μm (e.g., 10 μm, 5 to 10 μm, 10 to 15 μm).

40. The method of any one of embodiments 34-39, wherein tight spheroid cell aggregates are formed after about 48 hours of culture.

41. The method of any one of embodiments 34-40, wherein the cell aggregate is elongated for a period of 7-12 days, e.g., about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, and about 12 days.

42. The method of any of embodiments 34-41, wherein the number of cells used for seeding is about 200-1000 cells, e.g., 300 cells, 400 cells, 500 cells, 600 cells, 700 cells, 800 cells, 900 cells and 1000 cells (preferably about 400 cells).

43. A somite structure produced by the method of any one of embodiments 34-42.

44. The somite structure of embodiment 43, wherein the structure mimics an elongated embryonic tail bud and constructs the anterior-posterior axis.

45. A composition comprising the somite pre-mesodermal progenitor cells of embodiment 13 or 21 or the somite structure of embodiment 44.

46. Use of the somite structure of embodiment 44 in modeling human disease of the mesodermal lineage.

47. Use of the somite structure of embodiment 44 in the screening of a drug, including small molecule, protein, and antibody-based therapeutics.

48. Use of the somite anterior mesodermal progenitor cells of embodiment 13 or 21 or the composition of embodiment 45 in the treatment of a disease or disorder in a subject in need of muscle regeneration, cartilage regeneration, spinal cord regeneration or related regeneration.

49. A method for treating a disease or disorder requiring muscle regeneration, cartilage regeneration, and/or spinal cord regeneration in a subject, comprising administering (e.g., implanting) the somite anterior mesodermal progenitor cells of embodiment 13 or 21 or the somite structure of embodiment 45 to the subject.

50. A kit for reprogramming urine cells to somite anterior mesodermal progenitor cells, comprising:

a basal medium which is Advanced DMEM/F12;

combinations of agents for inducing urine cells, including CHIR99021, EPZ5676, bFGF and EGF, or CHIR9902, a8301, bFGF and EGF; and

optionally, means for collecting urine cells or collecting epithelial-like cells.

51. A kit for differentiating the somite pre-mesodermal progenitor cells of embodiment 13 or 21, comprising:

A basal medium which is DMEM/F12 or DMEM; and

a combination of agents selected from the group consisting of:

(a) KSR, ITS, NEAA, beta-ME, IGF-1, HGF, CHIR99021, VC, dex and SB431542;

(b) KSR, horse serum, NEAA, beta-ME, IGF-1 and HGF;

(c) FBS, VC, beta-glycerophosphate and 1-thioglycerol; and

(d) FBS, ITS, sodium pyruvate, VC, beta-glycerophosphate, TGF-beta 3 and BMP2.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings.

Brief Description of Drawings

Fig. 1 shows the formation of uifsm.

FIG. 1a shows a schematic diagram of the central nervous system of a vertebrate embryo, demonstrating the population of cells that produce the Central Nervous System (CNS). The posterior spinal cord is derived from neuromesodermal progenitor cells (NMp; red/green) which are located in the anterior primordial stripe (PS; brown) and adjacent caudal lateral ectoderm (CLE; light gray). NMp produces neural progenitor cells (Np; green) and contributes to CLE (light gray) then neural tubes (PNT; dark gray), and new mesodermal progenitor cells (Mp; red) which contribute to anterior mesoderm of the somite (PSM; brown) and then produces somites.

Fig. 1b shows a schematic diagram of uifsm reprogrammed from UC. UC, human-derived urine cells; uiPSM, somite anterior mesodermal progenitor cells induced by human-derived urine cells. Representative figures show the morphological changes of cells throughout UiPSM reprogramming. Scale bar, 100 μm.

FIG. 1c shows immunofluorescent staining by UiPSM reprogramming day 9, MIXL1 (red) with T (green) (upper panel), CDX2 (red) with TBX6 (green) (middle panel), SALL4 (red) with SOX2 (green) (lower panel). Scale bar, 200 μm.

FIG. 1d shows the expression of PSM-specific genes T, MIXL, TBX6 and CDX2 throughout the reprogramming process. qRT-PCR analysis was performed on layer-related genes during the entire reprogramming process of UiPSM, relative to DE (definitive endoderm), NSC (neural stem cells) and hESC (human embryonic stem cells), respectively. Endoderm-related genes are SOX17 and FOXA2; ectoderm-related genes are SOX1 and PAX6; the pluripotency-related genes are SOX1 and PAX6; expression levels were normalized to GAPDH. Data are mean ± standard deviation, n=3 independent experiments, p < 0.001.

Figure 1e shows a representative flow assay for the inducible efficient assessment of T protein expression throughout uifsm reprogramming. HPS (hESC-induced primordial streaks) served as positive control for T-positive cells. The expression of the pluripotency representative protein NANOG during UiPSM reprogramming was also examined.

Figure 1f shows t-SNE distribution of all 32798 cells on day 0, day 3, day 6 and day 9 during the entire uifsm reprogramming process, with cells at different time points being stained differently.

FIG. 1g shows marker gene expression patterns over t-SNE distribution. PSM markers (CDX 1, CDX2, DLL3, HES7, MIXL1, LEF1, TBX6, WNT5B, HOXB1, FGF8, T, MSX 1) were highly expressed on day 9, renal epithelial markers (PAX 8, EPCAM, AHI 1) were enriched on day 0, were low expressed on day 3, and protein synthesis-related genes (CD 44, FN1, ITGA 3) were mainly expressed during the processes including day 3 and day 6.

FIG. 1h shows the annotation analysis of the enriched genes for each cluster. The P value is less than 0.05.

Figure 2 shows stable in vitro expansion of uifsm colonies.

FIG. 2a shows representative images of the generation of UiPSM colonies of different passage numbers stably cultured in DM medium. Scale bar, 100 μm. DM: uifsm maintenance medium.

FIG. 2b shows immunofluorescence results of UiPSM colonies of different passage numbers, with MIXL1 (red) co-stained with T (green) on the left and TBX6 (green) co-stained with CDX2 (red) on the right. The scale bar represents 200. Mu.m.

FIG. 2c shows the extranodal mesoderm signature gene expression (T, MIXL, TBX6, CDX 2) of three donor-derived UIPSM colonies.

Fig. 2d depicts growth curves for UC and uifsm. The cells were mixed at 1X 10 ⁵ Individual cells/wells (24-well plate) were seeded and passaged every 5 days. Data are mean ± standard deviation (error bars) (n=3 wells).

FIG. 2e shows a correlation analysis showing the similarity between UiPSM clones of different passage numbers and the differences between UC and UiPSM. UC-1# and UC-2# refer to UC obtained from donors 1 and 2, respectively, and UiPSM1 and UiPSM2 refer to UiPSM cells obtained by reprogramming UC-1#1 and UC-2#, respectively.

Figure 2f shows the GO-rich features and p-values for UiPSM colonies on days 9 and 18.

Figure 3 shows UiPSM colonies maintain mesodermal features in vivo.

Figure 3a shows representative images of differences in teratoma morphology and size formed by UiPSM and UiPSC clones, respectively, over a month.

Fig. 3b shows representative images of H & E staining of UiPSM and UiPSC cloned teratomas derived from urine cells. Panel a. Glandular epithelium (endoderm); panel b. osteoblasts (mesoderm); panel c. Pigment cells (ectoderm); panel d. chondrocytes (mesoderm); panel e. Osteoblasts, osteoclasts, bone cells (mesoderm); panel f muscle cells (ectoderm); scale bar, 100 μm.

Fig. 3c shows t-SNE distribution of all 12456 cells of uippsm and uippc clone teratomas derived from urine cells.

FIG. 3d shows the cluster-colored t-SNE distribution identified by the Louvain algorithm.

FIG. 3e shows the t-SNE plot (bottom) of the M1 cluster subpopulation of FIG. 3 d.

FIG. 3f shows GO analysis for each cluster-enriched gene in FIG. 3 d. P value <0.05.

FIG. 3g shows GO analysis of the enriched genes for each of the sub-clusters in FIG. 3 e. P value <0.05.

FIG. 4 shows in vitro differentiation of UIPSM into mature mesodermal cell types.

FIG. 4a shows a schematic representation of stepwise differentiation of skeletal muscle cells (SKM) from UiPSM. The lower panel shows morphological changes in differentiation from UiPSM cells to skeletal muscle filaments. Scale bar, 100 μm.

FIG. 4b shows representative gene expression of human skeletal muscle satellite cells (PAX 7) and skeletal muscle cells (MYOD, MYOG, MRF, MYH3, MYH 7) during differentiation. Data are mean ± standard deviation, n=3 independent experiments.

FIG. 4c shows immunofluorescence of MYOD, MF20, DESMIN, LAMIN at day 60 of differentiation of skeletal muscle cells from UIPSM. Scale bar, 100 μm. The bottom value represents the percentage of statistically positive cells.

Fig. 4d shows the enriched GO term for UiPSM and differentiated skeletal muscle cells on days 30 and 60.

Fig. 4e shows a schematic representation of chondroblasts Alcin blue staining of chondroblasts differentiated from UiPSM on day 15. Scale bar, 100 μm.

FIG. 4f shows representative gene expression of chondroblasts during differentiation, including ACAN, COL9A1, SOX9, COL2A1. Data are mean ± standard deviation, n=3 independent experiments.

Fig. 4g shows representative GO enrichment features in UiPSM and chondroblasts based on up-regulated genes in each sample.

FIG. 4h shows a schematic representation of the differentiation of UIPSM into osteoblasts. Alizarin red staining of osteoblasts on day 15. Scale bar, 100 μm.

FIG. 4i shows representative gene expression of osteoblasts during differentiation, including OCN, SP7, BMP2, RUNX2. Data are mean ± standard deviation, n=3 independent experiments.

Fig. 4j shows representative GO enrichment terminology in UiPSM and osteoblasts based on up-regulated genes in each sample.

Fig. 5 shows UiSomitoid self-assembled into UiSomitoid with anterior and posterior axes and established molecular clocks for embryogenic somites.

Figure 5a shows a schematic representation of UiSomitoids generated from UiPSM by treatment extending for 7 days after aggregation for 2 days. The lower panel shows the extension of UiSomitoids. Scale bar, 200 μm.

FIG. 5b shows immunofluorescence of T (red) and CDX2 (red) co-stained with SOX2 (green) on day 9 in UiSomitoids, respectively. The scale bar represents 200. Mu.m.

FIG. 5c shows that the fluorescence signal of 8 peppers was measured with the Incucyte S3 living cell analysis system, converted to a digital signal by ImageJ, and then normalized to the maximum oscillation peak. Finally, an oscillation trend line was plotted on Prism 8 using a "zero-less baseline sine wave". The period is calculated as the peak-to-peak average (n > 10).

Fig. 5d shows statistical periodic changes in HES7 and MESP2 reporters produced by recording the changes in the parameter "wavelength" in "fig. 5 c".

FIG. 5e shows the statistical amplitude changes of HES7 and MESP2 reporter genes normalized to baseline by recording the changes in the parameter "amplitude" in "FIG. 5 c".

Fig. 5f is a schematic representation of somite occurrence in a mammalian embryo. Deep yellow, FGT/WNT signaling concentration distribution region; pale yellow, densely distributed areas of RA/BMP signaling; green, dynamic expression of HES 7. The bottom image shows the elongation of uiSomitoids along the A-P axis (green HES 7-GFP). Scale bar, 400 μm.

FIG. 5g shows the front-back profile of the PSM backend trait genes (T, SOX and CDX 2) in UiSomitoid.

FIG. 5h shows a transcriptome analysis of Geo-seq, 1500 highly variant genes were selected by calculating the variance of the UiSomitoid front and back axes. And classifying the screened genes into 6 groups by adopting a fuzzy clustering method.

FIG. 5i shows the gene trend along the front-rear axis of selected gene clusters in UiSomitoid and corresponding enriched GO features.

Detailed Description

All publications cited in this specification are herein incorporated by reference as if fully set forth. If certain content of the references cited herein contradict or are inconsistent with the present disclosure, the present disclosure controls.

For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific descriptions will be used. However, it should be understood that the scope of the present disclosure is not intended to be limited thereby.

In the present disclosure, unless otherwise specified, scientific and technical terms used herein have meanings commonly understood by those of skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the entire specification.

As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

It is to be understood that this disclosure is not limited to the particular methods, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art.

Unless the context requires otherwise, the terms "comprise," "include," and "contain" or the like are intended to imply the inclusion of a non-exclusive inclusion, such that a list of elements or features listed or not only those elements but also other elements or features not listed or stated.

The term "about" as used herein with respect to a stated value or range of values is meant to encompass variations of the stated value or range of values (i.e., to mean a little more or less than the stated value or range of values, within + -20%, + -10%, + -5%, + -1%, + -0.5%, + -0.1% or + -one standard deviation range of the stated value or range of values). Although efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, concentrations, etc.), some experimental deviation should be accounted for.

Unless otherwise indicated, percentages of concentrations provided herein are by weight. For example, 1% ITS or 1% sodium pyruvate refers to the weight percent of ITS or sodium pyruvate in the prepared medium.

Definition of the definition

As used herein, the term "somite anterior mesoderm" refers to the region of the mesoderm of a neural embryo that is flanking and formed simultaneously with a neural tube. Cells in this region produce somites, i.e., pieces of tissue that extend along both sides of the nerve tubes, forming muscles and back tissue, including connective tissue and dermis.

As used herein, the term "anterior-mesodermal (PSM) cells" are precursors of the somites, which flank the nerve tubes and create the musculoskeletal system that shapes the body of vertebrates. WNT and FGF signaling have been found to control PSM and somite formation and to exhibit a characteristic of anteroposterior axis differential distribution in UiSomitoid. The ability to efficiently produce PSM cells in vitro may facilitate the study of a gene regulatory network that controls the formation of nascent PSM cells and their conversion to differentiation/paraxial mesoderm.

As used herein, the term "mesodermal progenitor cells" refers to a population of undifferentiated progenitor cells derived from early gastrulation. At the end of gastrulation, they have moved to the very end of the embryo, a region called the tail bud. During whole gastrulation and somite development, mesodermal progenitor cells continue to contribute cells to a site predetermined as the pre-somite mesoderm, using complex molecular clocks and wavefront mechanisms to generate species-specific numbers of segments to segment the pre-somite mesoderm (reviewed in Tam et al Current Top Dev biol 2000,47:1-32; holley and Takeda, semin Cell Dev biol 2002Dec,13 (6): 481-8; dubrulle and Pourquie, development.2004Dec,131 (23): 5783-93).

As used herein, the terms "reprogramming," "inducing," and grammatical equivalents thereof refer to the process of altering or reversing the differentiation state of partially or terminally differentiated somatic cells (e.g., urine cells). Reprogramming of somatic cells may be a partial or complete reversal of the state of somatic cell differentiation. In some embodiments, when a somatic cell is reprogrammed to induce pluripotent stem cells, the reprogramming is complete. In some preferred embodiments, reprogramming is partial, e.g., reverting to any less differentiated state. For example, terminally differentiated cells are reversed into less differentiated cells, such as pluripotent cells, progenitor cells (e.g., somite anterior mesodermal progenitor cells). The reprogramming process may be caused by induction of somatic cells with a set of small molecule compounds (e.g., WNT agonists, DOT1L inhibitors, and growth factors), or by expression of transcription factors and micrornas in somatic cells (e.g., POU5F1, KLF4, SOX2, c-MYC, and MIR302-367 clusters). For example, reprogramming urine cells to somite anterior mesodermal progenitor cells can be achieved by induction with a specific combination of factors and compounds, as disclosed herein.

As used herein, the term "GO" or "gene ontology" is available from Gene Ontology Consortium and generally consists of a set of classes (or terms or concepts) between which there are relationships. Gene ontology can be used to describe the role of genes and gene products in all organisms. Gene ontologies can characterize the relationship between genes and keywords assigned to each gene. For example biological processes, cellular components and molecular functions of gene ontology terms can be found in http:// www.geneontology.org., can use for example EASE software for GO analysis.

As used herein, the term "passaging" refers to the step of detaching cells from their support (by enzyme or enzyme mixture) and diluting the cells in culture medium before inoculating the cells to a new support for growth. For example, "p9" herein refers to cells that have been passaged 9 times.

Isolation and expansion of urine cells

The cells used as starting material in the reprogramming or induction herein are urine cells of human origin. The present disclosure shows that urine cells are an ideal source of functional lineage specific cells in terms of favorable genetic profiles and inherent multipotent potential. Unlike the production of urine stem cells from urine cells, the production of functional lineage specific progenitor cells can exit the pluripotent state, reducing the risk of tumor formation, and thus has broad application prospects in clinical treatment. As disclosed herein, reprogramming human urine cells successfully produces extraganglionic mesodermal progenitor cells (uipsms) that can be further differentiated into extraganglionic mesodermal lineage cells (e.g., muscle cells and bone cells).

Urine samples can be collected from a subject in a simple and non-invasive manner. Urine cells can be isolated from urine samples using cost-effective and simple isolation methods. Non-invasive and easy isolation are major advantages of urine-derived cells compared to all other donor-related samples. Adipose derived stem cells, hair cells, fibroblasts, amniotic cells, skin epithelial stem cells, proximal tubule (HK 2) cell lines, pericyte derived cells, endothelial cells, exopancreatic secretory cells and mesenchymal stromal cells require liposuction or invasive methods for sample isolation; amniotic fluid and umbilical cord cells are neither readily available nor suitable for autograft. Instead, urine cells can be pelleted by simple sample centrifugation and isolated by culturing and expanding the cells in a cell growth medium (e.g., REGM medium). The cell culture medium may be selected to promote the growth of certain types of urine cells. In some embodiments, the growth medium used to expand urine cells promotes the growth and viability of epithelial (epithelial-like) cells, and thus may be enriched for epithelial-like and mesenchymal cell types during expansion.

When collecting adherent urine cells grown into larger masses on a culture plate, the epithelial-like cells can be harvested with a tool (e.g., a long glass pipette) or rapidly digested with 0.25% trypsin-EDTA on a new culture plate (this approach is because the epithelial-like cells are more digestible than mesenchymal cells), thereby obtaining the epithelial-like cells.

Epithelial-like urine cells induce generation of somite anterior mesodermal progenitor cells (UiPSM)

In some embodiments, epithelial-like cells enriched in urine cell culture are selected for further induction. Preferably, prior to induction, the epithelial-like cells are pretreated by electroporation with a vector (e.g., pEP4EO2SET2K and pCEP 4-miR-302-367) to sensitize the cells to induction. pEP4EO2SET2K is a non-integrated episomal vector encoding OCT4, SOX2, SV40LT, KLF4, pCEP4-miR-302-367 is a non-integrated episomal vector encoding miR302-367 cluster, useful for promoting cell permeability.

For induction, the epithelial-like cells may be incubated in basal medium supplemented with a selected set of compounds and molecules. In some embodiments, urine-derived epithelial-like cells are induced in basal medium supplemented with WNT agonist CHIR99021, DOT1 inhibitor EPZ5676 and one or more growth factors selected from FGF (e.g., bFGF), EGF, VEGF, PDGF, TGF- β, PD-ECGF, TNF, HGF, IGF (e.g., IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof. Specifically, the basal medium may be supplemented with bFGF and EGF. In some embodiments, the basal medium is Advanced DMEM/F12. In some further embodiments, the basal medium is supplemented with CHIR99021 in the 2-4 μM concentration range (e.g., 3 μM), EPZ5676 in the 4-6 μM concentration range (e.g., 5 μM), bFGF in the 4-6ng/μL concentration range (e.g., 5ng/μL), and EGF in the 4-6ng/μL concentration range (e.g., 5ng/μL).

Induction may last for about 7 to 12 days until epithelial-like cells are observed to bind tightly together to form clones. In this process, morphological, biological and molecular changes that induce cells to occur at different time points can be detected by various methods and assays. The obtained granular colony is morphologically similar to human stem cells (primer stem cells) to be activated and naive stem cellsstem cells) or NPCs are dissimilar. These reprogrammed or induced cells were designated UiPSM cells (urine-derived somite anterior mesodermal progenitor cells).

Several markers are known to be associated with anterior mesodermal cells of the somites, including T (also known as Brachury and TBXT), mix 1, TBX6 and CDX2. Expression of the markers and their changes can be determined by established methods, such as Q-PCR, immunofluorescence, sequencing of batch and single cell RNA-seq, FACS, etc. The inventors have demonstrated that, during induction as described above, somite anterior mesoderm-related markers are progressively expressed. In contrast, multipotent markers (such as POU5F1 and NANOG), endodermal markers (SOX 17 and FOXA 2), or ectodermal markers (such as SOX1 and PAX 6) were hardly expressed. Thus, no pluripotency is required during reprogramming, and the resulting somite anterior mesodermal progenitor cells are not committed to endodermal and ectodermal features, reducing the risk of tumor formation in vivo.

Tables a and B below describe genes that are highly expressed and lowly expressed in UiPSM cells, respectively.

Table A

Table B

In vitro expansion of UiPSM cells

Reprogrammed UiPSM cells are further characterized in that they can proliferate stably and rapidly in vitro. In some embodiments, uiPSM cells are cultured or passaged in defined media that can maintain stable amplification of UiPSM. In some embodiments, the defined medium comprises WNT agonist CHIR99021, TGF- β inhibitor a8301 and one or more growth factors selected from the group consisting of: FGF (e.g., bFGF), EGF, VEGF, PDGF, TGF-beta, PD-ECGF, TNF, HGF, IGF (e.g., IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof. Specifically, the growth factors selected may be bFGF and EGF. In some embodiments, the basal medium is Advanced DMEM/F12. In some further embodiments, the defined medium comprises CHIR99021 in the concentration range of 2-4. Mu.M (e.g., 3. Mu.M), A8301 in the concentration range of 4-6. Mu.M (e.g., 5. Mu.M), bFGF in the concentration range of 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l), and EGF in the concentration range of 4-6 ng/. Mu.l (e.g., 5 ng/. Mu.l).

As used herein, a concentration range of 2-4 μm refers to a range of approximately 2-4 μm and includes any value and subrange within the range, such as 2 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4 μm. Similarly, a concentration range of 4-6 μΜ is meant to be approximately a range of 4-6 μΜ and includes any values and subranges subsumed therein, such as 4 μΜ, 4.2 μΜ, 4.4 μΜ, 4.6 μΜ, 4.8 μΜ, 5 μΜ, 5.2 μΜ, 5.4 μΜ, 5.6 μΜ, 5.8 μΜ, 6 μΜ. A concentration range of 5-15. Mu.M refers to a range of approximately 5-15. Mu.M and includes any value and subrange within the range, such as 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, 9. Mu.M, 10. Mu.M, 11. Mu.M, 12. Mu.M, 13. Mu.M, 14. Mu.M, 15. Mu.M; a concentration range of 4-6 ng/. Mu.l refers to a range of approximately 4-6 ng/. Mu.l and includes any value and subrange encompassed within the range, such as 4ng/ul, 4.2 ng/. Mu.l, 4.4 ng/. Mu.I, 4.6 ng/. Mu.I, 4.8 ng/. Mu.l, 5 ng/. Mu.l, 5.2 ng/. Mu.l, 5.4 ng/. Mu.l, 5.6 ng/. Mu.l, 5.8 ng/. Mu.l, and 6 ng/. Mu.l.

The passaged UiPSM cells maintained their properties as the first generation UiPSM induced from urine cells. qRT-PCR, immunofluorescence and RNA-seq demonstrated that serial passage UiPSM continued to stabilize expression of anterior-ganglion mesoderm signature markers T, MIXL, TBX6 and CDX2.

Differentiation of somite anterior mesodermal progenitor cells (UiPSM)

The resulting somite anterior mesodermal progenitor cells (uipsms) have specific stem properties because of their ability to self-renew and differentiate specifically into somite anterior mesodermal lineage cells in various selected optimal media. The somite anterior mesodermal progenitor cells (UiPSM) can be successfully differentiated in vitro into osteoblasts, chondroblasts and skeletal muscle cells. UiPSM cells obtained by the methods of the present disclosure may be cultured in vitro under differentiation conditions to produce the desired cells. Various methods of differentiation are known in the art (see Zhou et al, generation of human induced pluripotent stem cells from urine samples, nat Protoc 7,2080-2089,2012; beccari et al, multi-axial self-organization properties of mouse embryonic stem cells into architecture 562,272-276,2018; lee et al, mesenchymal stem Cell-conditioned medium enhances osteogenic and chondrogenic differentiation of human embryonic stem cells and human induced pluripotent stem cells by mesodermal lineage reduction, tissue Eng Part A20,1306-1313, 2014; nejadnik et al, improved approach for chondrogenic differentiation of human induced pluripotent stem cells, stem Cell Rev Rep 11,242-253,2015; shelton et al, derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells, stem Cell Reports 3,516-529, 2014) which may be used to differentiate UIPSM cells herein. Preferably, an optimized differentiation protocol for different target cells may be used. As demonstrated in the working examples, uiPSM cells have the ability to differentiate into muscle cells, osteoblasts, bone cells and chondrocytes, respectively, under defined conditions. Q-PCR, immunofluorescence, FACS and bulk RNA-seq data support the successful establishment of the self-renewing and pre-somite lineage specific differentiation system described above.

In some embodiments, skeletal muscle cells differentiated from somite anterior mesodermal progenitor cells (UiPSM) have a gene expression profile similar to naturally occurring skeletal muscle cells and are involved in muscle regeneration. As used herein, a "similar expression profile" with respect to UiPSM cells refers to a relative expression profile of markers in UiPSM cells of the present disclosure that is similar to the relative expression profile of published naturally occurring somite anterior mesodermal cells.

In some embodiments, the osteoblasts or bone cells differentiated from the somite pre-mesodermal progenitor cells have a gene expression profile similar to that of naturally occurring osteoblasts or bone cells.

In some embodiments, the chondrocytes differentiated from the somite pre-mesodermal progenitor cells have a gene expression profile similar to that of naturally occurring chondrocytes.

Urine-derived somite anterior mesodermal progenitor cells self-assemble to form UiSomitoid

Somite anterior mesodermal progenitor cells (uipsms) have the ability to self-assemble in vitro to form specific somite-like structures (referred to herein as uisomitoids). The inventors have surprisingly found that a certain number of UiPSM cells (e.g., about 200-600, about 300-500, about 400 UiPSM cells) can aggregate, expand and elongate into a 3D structure. The process is preferably performed first in a defined medium to form tight spherical aggregates, and then elongating the aggregates in a basal medium supplemented with WNT agonist and NODAL inhibitor. In some embodiments, the defined medium is Advanced DMEM/F12 supplemented with CHIR99021 in a concentration range of about 2-4. Mu.M (e.g., 3. Mu.M, 2.5-3. Mu.M, 3-3.5. Mu.M), A8301 in a concentration range of about 4-6. Mu.M (e.g., 5. Mu.M, 4.5-5. Mu.M, 5-5.5. Mu.M), bFGF in a concentration range of about 4-6 ng/M. Mu.l (e.g., 5 ng/. Mu.l, 4.5-5 ng/mu.l, 5-5.5 ng/mu.l) and EGF in a concentration range of about 4-6 ng/mu.l (e.g., 5 ng/mu.l, 4.5-5 ng/mu.l). In some embodiments, the basal medium for elongation is supplemented with WNT agonist CHIR99021 and NODAL inhibitor SB431542. In some embodiments, basal medium is supplemented with WNT agonist CHIR99021 in the concentration range of 2-4. Mu.M (e.g., 3. Mu.M) and NODAL inhibitor SB431542 in the concentration range of 5-15. Mu.M (e.g., 10. Mu.M). This 3D structure, known as UiSomitoid, shows a "coracoid" cell-dense region and polar extension toward the "caudal" end, resembling an elongated embryo tail bud. UiSomitoid has a "coracoid tail" structure with anterior-posterior axes that can be used to simulate the molecular clock of embryogenic body segments.

In addition to the medium components, optimization of the UiSomitoid production program may also take into account other factors such as cell density, BMP inhibitors, nodal signaling during aggregation and elongation steps, and the like.

The UiSomitoid structures provided herein show polarized expression patterns of SOX2/CDX2, SOX2/T and CDX2/T in paired co-staining with antibodies, indicating the presence of anterior and posterior tissues. Geo-seq data analysis supports UiSomitoid to successfully build the front and back axes. UiPSM can self-organize into a "coracoid tail" structure (Somitoid) with anterior-posterior axis and establish the molecular clock at which embryoid body segments occur.

Urine cell-derived uiSomitoid constructs

In recent years, many 3D-like embryos have been produced from Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs), and other Pluripotent Stem Cells (PSCs) that mimic various stages of early embryo development (Li et al, 2019; matthews et al, 2021; yu et al, 2021). Mammalian ESCs and human ESCs are induced to produce gastrulations similar to those of an embryo in development of the gastrulation stage (Beccari et al, 2018; moris et al, 2020;van den Brink et al, 2014). These classes of embryos allow for testing and improvement of theory and hypothesis, can be easily manipulated, genetically manipulated, or generated in various ways to improve experimental design, and allow for detailed observation of cellular events in real time when combined with fluorescent reporter constructs. These species of embryos can also be produced in large quantities for statistical analysis, which is difficult to perform on human embryos due to capital constraints and ethical issues (Matthews et al, 2021).

The most convincing evidence for dual fate neuromesodermal progenitor (NMP) comes from the tail bud cell population with self-renewing properties, which contribute to both spinal cord and paraxial mesoderm. Paraxial mesoderm (also known as somite anterior mesoderm) cells can develop into skeletal muscle lineages, muscle cell lineages, and dermis cell lineages, which further differentiate into musculoskeletal systems. Thus, the in vitro construction of self-renewing PSM progenitors is important for somatical studies, where it is difficult to obtain self-renewing PSM cells in human embryos due to ethical and technical limitations.

Although human embryonic stem cells (ES) can differentiate into PSM and generate a three-dimensional "gastrulation" model, studies of somite generation are more complex due to the gastrulation process. Furthermore, ipscs have the potential to differentiate into germ layers, while their risk of tumorigenicity limits the application. Thus, cells induced by iPSC may also have a tumorigenic risk, as the induction efficiency cannot reach 100%. In addition, a variety of cells including fibroblasts, blood cells, amniotic cells, skin epithelial stem cells, proximal tubule (HK 2) cell lines, pericyte-derived cells, endothelial cells, exocrine pancreatic cells are sources of initiation of reprogramming induction, and the acquisition of these cells may damage the subject to some extent. Furthermore, urine cells obtained non-invasively can be reprogrammed to iPSC and NPC, indicating that urine cells are a good source of reprogramming.

As disclosed herein, the UiSomoid structure is a self-organizing and self-renewing structure that is characterized by the development of segments, including the establishment of the anterior-posterior axis and molecular clocks of embryoid body segments. Thus, the UiSomitoid structure is a promising tool for studying the occurrence of somites.

Advantageous technical effects

The invention provides a UiPSM reprogramming system which is efficiently carried out from urine cells, and the established UiPSM cells highly express related genes of anterior mesoderm of somites including T, MIXL, TBX6 and CDX2 and construct a transcriptional characteristic expression profile of anterior mesoderm of somites. In addition, the UiPSM cells produced do not have the indicated multipotent, endodermal and ectodermal features and reduce the risk of tumor formation in vivo.

UiPSM colonies can be stably amplified in vitro and have specific differentiation potential in vivo or in vitro. In addition, uiPSM-derived skeletal muscle cells can survive and participate in muscle regeneration.

Notably, stably amplified UiPSM colonies can self-organize into a "head-to-tail" structure (UiSomitoid) with front-to-back axes and mimic molecular clock oscillations during embryogenic body-segment generation.

In summary, the inventors established self-renewing uipmsm cells reprogrammed from urine cells that can differentiate into different somite anterior-mesodermal lineage cells and self-assemble in vitro into a "head-tail" structure (UiSomitoid) with anterior-posterior axis characteristics, mimicking somite generation.

UiPSM and UiSomitoid application

UiPSM obtained or obtainable from the methods of the present disclosure may advantageously be cultured in vitro under differentiation conditions to produce differentiated cells, particularly cells of the somite anterior mesodermal lineage, such as skeletal muscle cells, osteoblasts, chondrocytes and chondroblasts, muscle, cartilage, bone, dermal tissue, and the like.

The skilled artisan can use known protocols for differentiating stem cells, such as those conventionally used to differentiate induced pluripotent stem cells, ES cells, or mesenchymal stem cells into a desired cell line. Preferably, these schemes can be optimized based on specific requirements.

One of the main fields of application of UiPSM and UiSomitoid is cell therapy or regenerative medicine.

For example, according to the methods of the present disclosure, urine cells obtained from a subject can be cultured, subsequently reprogrammed to UiPSM, and differentiated into a suitable cell line for administration to the subject, e.g., the same subject as the cell donor (autologous treatment). Similarly, regenerative medicine can be used to potentially cure any disease caused by dysfunction, damage or failure of muscle or bone tissue by directly implanting in vivo a composition comprising UiPSM or a derivative thereof (comprising appropriate progenitor cells or cell lineages) to regenerate damaged tissue in vivo.

In one aspect, the reprogrammed uifsm may be used for autologous regenerative therapy in patients in need of regenerative therapy for particular disorders or therapies associated with such disorders, including but not limited to muscle and bone disorders, neurological disorders, and other metabolic disorders. In another specific embodiment, the reprogrammed UiPSM composition is for use in treating a joint or cartilage, muscle or bone injury.

In another specific embodiment, uiPSM and UiSomitoid may also be advantageously used to generate, for example, but not limited to, dermal, muscle or skeletal cells from healthy or diseased patients for screening applications in the pharmaceutical industry. Such screening tests may be used to search for new drugs or toxicology tests with clinical application. In another embodiment, uiPSM and UiSomitoid may also be used to regenerate bone tissue.

In another specific embodiment, reprogrammed UiPSM and UiSomitoids may also be used to regenerate neuronal tissue, for example in patients with neurodegenerative diseases.

According to some embodiments, a method for treating a condition may comprise transplanting or implanting a UiPSM cell population or UiSomitoid (e.g., those described above) onto or into a tissue or organ of a subject. "treating" or "treatment" of a condition may refer to preventing the condition, lessening the severity of the condition, slowing the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the progression of symptoms associated with the condition, reducing or ending the symptoms associated with the condition, such that the condition completely or partially subsides, or some combination of these aspects. According to embodiments described herein, the treatment of a condition may involve implantation or transplantation of UiPSM cell populations and uisomitoids. The appropriate implantation or implantation method may be selected depending on the location (e.g., what type of tissue or organ) the cell population is to be implanted or implanted. For example, implantable or injectable grafts may be used to treat a condition.

The implantable graft may include a solid matrix that allows UiPSM cells to be seeded with the necessary growth factors (e.g., the particular environment in which the cell population is to be implanted), cultured, and then implanted into the tissue or organ of the subject having the condition. The injectable implant may fill any defective shape or space in the damaged organ or tissue. The injectable graft comprises injecting UiPSM cells or reprogrammed somatic cells into a cell suspension containing biological material that is cured in situ by various crosslinking methods known in the art. The mixture may be injected directly into the tissue or organ or may be exposed or adhered to the surface of the tissue or organ.

Non-limiting examples of biological materials that can be used in the injectable implant include, but are not limited to, inorganic materials, natural materials such as chitosan, alginate, hyaluronic acid, fibrin, gelatin, and many synthetic polymers. Such materials are typically cured by thermal gelation, photocrosslinking, or chemical crosslinking. The cell suspension may also be supplemented with soluble signals or specific matrix components. Because these grafts can be relatively easily injected into the target area, invasive surgery is not required (or is minimally required), which reduces costs, patient discomfort, risk of infection, and scar formation. Chemically modified HA is also useful as an injectable material for tissue engineering because it HAs a durable effect while maintaining biocompatibility. The cross-linking method also maintains the biocompatibility of the material, its presence in a wide range of regenerative or stem/progenitor niches makes it an attractive injectable material.

Kit for detecting a substance in a sample

In one aspect, the invention relates to a kit for inducing reprogramming of urine cells, comprising:

a) Agents for reconstitution of urine cell-inducing medium, including Advanced DMEM/F12, CHIR99021, EPZ5676, bFGF and EGF;

b) Optionally, means for collecting urine cells;

c) Optionally, means for picking up epithelial-like cells, such as a long glass pipette.

In another aspect, provided herein is a kit for rational drug design comprising reprogrammed cells obtained by the methods of the invention and cells derived or differentiated therefrom. In one embodiment, the kit includes UiSomitoid and instructions for its use in drug screening in a disease model. In another embodiment, the kit comprises UiPSM cells, medium for cell expansion, and instructions for use thereof.

Examples

The present disclosure will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure. These examples are not intended to be representative of all or only the experiments that follow.

Example 1: reprogramming urine cells to urine-derived somite anterior mesodermal progenitor cells (UiPSM)

1.1 vector preparation

pEP4-EO2S-ET2K plasmid was purchased from Addgene (catalog number: 20927). The plasmid contained the pCEP4 backbone and expressed Oct4, sox2, SV40LT and Klf4. The pCEP4-miR-302-367 plasmid was prepared by incorporating microRNA clusters 302-367 into the pCEP4 backbone (preparation as described by Liao et al, microRNA cluster302-367enhances somatic cell reprogramming by accelerating a mesenchymal-to-epihelian transformation. J Biol Chem 286,17359-17364,2011; wang et al, generation of integration-free neural progenitor cells from cells in human, url. Nat Methods 10,84-89,2013).

1.2 reprogramming of urine cells

With informed consent, an upper urinary tract urine sample was collected from healthy subjects. Viable Urine Cells (UC) were isolated from urine samples and cultured with REGM (Lonza, CC-3190), which is commonly used to grow nephron-derived epithelial cells, including cells shed by nephrons during urine formation. Then, epithelial-like cells were picked up with a long glass pipette for subsequent experiments. Next, these epithelial-like cells (exceeding 1.5x10) were electroporated by the vectors pEP4EO2SET2K (6 μg) and pCEP4-miR-302-367 (4 μg) ⁶ Individual cells) were pretreated to make the cells sensitive to induction, and then cultured on REGM medium for two days to restore their ability (day 0 in fig. 1 b). Subsequently, in the presence of a WNT agonist CHIR99021 (3. Mu.M; internal synthesis), DOT1L inhibitor EPZ5676 (5. Mu.M; selleck Chemicals, S7062), bFGF (5 ng/. Mu.l; peproTech, P09038) and EGF (5 ng/. Mu.l; R) &Pretreated epithelial-like cells were induced in basal medium (Advanced DMEM/F12) from D systems,236-EG for 9 days.

We observed that successfully reprogrammed epithelial-like cells were tightly bound together to form clones (fig. 1b, day 3). After about 9 days, some granular colonies that were morphologically dissimilar to human Naive stem cells (primer stem cells) and Naive stem cells (Naive stem cells) or NPCs appeared, and these colonies were picked with a long glass rod pipette to establish a cell line (fig. 1b, day 9).

Q-PCR and immunofluorescence detected that this process gradually expressed the somite anterior-mesoderm (PSM) related markers, including T (also known as Brachury and TBXT), MIXL1, TBX6 and CDX2, while hardly expressing pluripotent markers such as POU5F1 and NANOG, endodermal markers SOX17 and FOXA2, or ectodermal markers SOX1 and PAX6 (FIGS. 1c and 1 d). The targeted endoderm cells (DE), neural Stem Cells (NSCs) and human embryonic stem cells (hescs) served as positive controls for the respective markers. FACS analysis showed that T expression reached 44% on day 9, but NANOG could not be detected throughout the process (fig. 1 e), supporting the lack of need for multipotent reprogramming during induction. We also analyzed single RNA-seq sequencing to transcriptionally characterize the entire reprogramming process, with the result that the reprogramming process was supported without establishing a pluripotency network (fig. 1f-1 h). These successfully reprogrammed cells are known as UiPSM cells.

1.3 in vitro continuous culture of UiPSM cells

Based on the conditions of CHIR99021 treatment, we optimized the induction medium and found an induction medium based on Advanced DMEM/F12 supplemented with the WNT agonist CHIR9902 (3. Mu.M), TGF-beta inhibitor A8301 (5. Mu.M; R)&D systems, 2939), bFGF (5 ng/. Mu.l) and EGF (5 ng/. Mu.l) in Defined Medium (DM) capable of maintaining stable amplification of UiPSM. This medium is also known as UiPSM maintenance medium or "DM medium". UiPSM cells were grown at 1X 10 ⁵ Individual cells/wells (24-well plate) were seeded and passaged every 5 days.

As shown in fig. 2a, uiPSM cells can be rapidly expanded in UiPSM maintenance medium. Similar to the above results, Q-PCR, immunofluorescence and batch RNA-seq sequencing data support passaged UiPSM stable expression of the anterior mesoderm trait genes. UiPSM of urine cells derived from donor 1, donor 2 and donor 3 were named UiPSM1, uiPSM2 and UiPSM3, respectively. Immunofluorescence analysis showed that serially passaged UiPSM cells (e.g., P1, P5, P9, P18) expressed T, MIXL, TBX6 and CDX2 (fig. 2 b), while barely expressed pluripotency markers such as POU5F1 and NANOG, endodermal markers SOX17 and FOXA2, or ectodermal markers SOX1 and PAX6. This result was also confirmed by Q-PCR analysis (FIG. 2 c). Comparison of urine cells and UiPSM is shown in figures 2d and 2 e. GO analysis of UiPSM colonies of P1, P9 and P18 at day 9 showed that UiPSM stably retained PSM characteristics such as somite development, segmentation and somite development during passage (fig. 2 f).

Example 2: differences between urine cell-derived PSCs (pluripotent stem cells, uiPSC) and PSM (somite anterior mesodermal progenitor cells, uiPSM)

The tumorigenic capacity of uiscs and uifsm was compared by teratoma experiments by forming teratomas in mice and examining representative tissues of all three germ layers. Uiscs reprogrammed from urine cells were obtained by following strictly published protocols (Zhou et al Generation of human induced pluripotent stem cells from urine samples. Nat Protoc 7,2080-2089,2012). Preparation of 3X10 embedded in Matrigel (diluted twice with DMEM/F12) separately ⁶ The uipmc (P6) or uipmc (P10) cells were subcutaneously injected on the backs of immunodeficient mice (MITRG mice, the Jackson Laboratory, 017711).

The size of uifsm-derived tissue blocks was significantly smaller than uifsc-derived tissue blocks (fig. 3 a). Paraffin embedding and HE staining were then performed (fig. 3b, panels a-c represent uiscs, panels d-f represent uifsm). The results indicate that UiPSM-derived tissues enrich for chondrocytes (fig. 3b, panel d), osteoblasts, osteoclasts, osteocytes (fig. 3b, panel e) and skeletal muscle cells (fig. 3b, panel f), which are mainly related to cells of the pre-somite mesodermal lineage. Urine cell-derived uipmcs enriched all endodermal, mesodermal and ectodermal lineage cells, indicating the potential to differentiate into cells of all three endodermal, mesodermal and ectodermal lineages, whereas uipms enriched only mesodermal lineage cells, indicating a decrease in "stem". The results indicate that UiPSM colonies reduce the risk of tumor formation.

In addition, tissue pieces of similar size derived from UiPSC at one month and UiPSM at two months were separately isolated, and single cell RNA-seq sequencing analysis was performed to analyze the cellular composition of tissues derived from UiPSC and UiPSM, respectively. GO profiling showed that UiPSM was significantly consistent with muscle development, further analysis of the sub-clusters, and that these sub-clusters were primarily involved in fate of the mesodermal lineage, such as morphogenesis of muscle, cartilage, bone, heart and limbs (fig. 3c-3 g), supporting the potential of UiPSM to have cells of the pre-somite mesodermal lineage, in contrast to UiPSC.

Example 3: assessment of UiPSM differentiation potential

Based on the differentiation protocols of skeletal muscle, osteoblast and chondrocyte of hES (Beccari et al, 2018; lee et al, 2014; nejadnik et al, 2015; shelton et al, 2014, supra), we modified different protocols to achieve induction of UiPSM to skeletal muscle, osteoblast and chondrocyte, respectively, in vitro. The optimization scheme is as follows:

for differentiation into skeletal muscle cells, uiPSM was digested into single cells using 0.25% trypsin-EDTA (Gibco, 25200056), and 7.5x10 ⁴ Individual cells were sparsely passaged into new Matrigel coated wells on 24-well plates overnight in UiPSM maintenance medium. For subsequent myogenic differentiation, uifsm was initially induced by an improved method comprising two stages. In the first stage, differentiation is carried out with defined SM medium for 15 days, the procedure being to replace the medium every 2 days; in the second stage differentiation was performed with defined DiKHI medium until skeletal muscle fiber bundles were completely present, which step replaced the medium every 3 days (FIG. 4 a).

SM: DMEM/F12 is supplemented with 15% KSR (Gibco, 10828028), 1% ITS (Gibco, 41400045), 1% NEAA, 0.1. Mu.M.beta. -ME, 4ng/ml IGF-1 (Pepro Tech, 250-19), 10ng/ml HGF (R & D systems, 294-HG-250), 3. Mu.M CHIR99021, 50ng/ml VC (Sigma-Aldrich, 49752), 0.5ng/ml Dex (Target Mol, T0947L) and 2nM SB431542.

DiKHI: DMEM was supplemented with 15% KSR, 2% horse serum (Gibco, 16050122), 1% NEAA, 0.1. Mu.M. Beta. -ME, 4ng/ml IGF-1 and 10ng/ml HGF.

For differentiation into osteoblasts, uiPSM was used at 7.5x10 ⁴ Individual cells were passaged overnight into new Matrigel coated wells on 24-well plates. DMEM was used as basal medium supplemented with 10% FBS (NTC, SFBE), 50ng/ml VC, 100nM beta-glycerophosphate (PeproTech, 154804-51-0) and 1. Mu.M 1-thioglycerol (Sigma, 96-27-5) for 15 days of UiPSM differentiation, with medium change every 3 days.

For differentiation into chondrocytes, uiPSM was used at 7.5x10 ⁴ Individual cells were passaged overnight into new Matrigel coated wells on 24-well plates. DMEM was used as basal medium supplemented with 10% FBS, 1% ITS, 1% sodium pyruvate (Gibco, 11360), 50ng/ml VC, 0.1nM beta-glycerophosphate, 4 ng-ml TGF-. Beta.3 (PeproTech, 100-36E) and 20ng/ml BMP2 (PeproTech, 500-P195) were used to differentiate UiPSM for 15 days with medium changes every 3 days.

Next, Q-PCR, immunofluorescence, FACS and RNA-seq in batches were performed separately to verify the differentiation system described above.

The 60 day-induced Q-PCR assay of skeletal muscle from UiPSM showed that mature muscle markers such as MYOD, MYOG, MRF, MYH3 and MYH7 were expressed at a much later stage (fig. 4 b), followed by immunostaining to track MYOD, MF20, desin and LAMIN on day 60. Immunofluorescence of MYOD, MF20, desin, LAMIN during differentiation of skeletal muscle cells from UiPSM on day 60 supported this result (fig. 4 c). GO profiling showed that cells produced on day 30 and day 60 were closely related to muscle tissue development and muscle contraction (fig. 4 d).

In addition, uiPSM can differentiate into chondroblasts and osteoblasts within 15 days (fig. 4e,4 h). Q-PCR confirmed this result by using markers for chondroblasts such as ACNA, COL2A1, SOX9, COL9A1 (FIG. 4 f) and markers for osteoblasts such as BMP2, RUNX2, BGLAP, SP7 (FIG. 4 i). Chondroblasts and osteoblasts can be further identified by staining with allrxin blue (GENMED, GMS 80015.1) and alizarin red (GENMED, GMS 90017.1). Consistently, RNA-seq data analysis also confirmed the identity of osteoblasts and chondroblasts based on the expression of the relevant genes (fig. 4g,4 j). Taken together, these results demonstrate that UiPSM, like the in vivo extranodal mesoderm, is capable of differentiating into skeletal muscle cells, osteoblasts and chondroblasts in vitro.

Example 4: generation of UiSometaid structures

About 400 UiPSM cells were seeded in wells of a low adhesion plate, showing that they can form tight spherical aggregates within 48 hours in Defined Medium (DM) and then extended in basal medium (Advanced DMEM/F12) called "CS medium" containing CHIR99021 (3 μm) and NODAL inhibitor SB431542 (10 μm). These aggregates gradually break symmetry, forming an elongated structure, and continue to grow over time, as measured by the longer (L) ultra-wide (W) axis, which shows a "coracoid" cell-dense region and polar extension toward the "caudal" end, reaching 1000 μm (L) on day 9, similar to an elongated embryo tail bud (fig. 5 a). This structure is called UiSomitoid, a urinary derived cell-induced somite-like structure.

The conditions for segment generation were further optimized by the following parameters: inhibitors of BMP, WNT, NODAL signaling during cell density, aggregation and elongation. Under optimized conditions, uiSomitoid showed polarized expression patterns of SOX2/CDX2, SOX2/T and CDX2/T when co-stained with antibody in pairs on day 9, indicating the presence of anterior and posterior tissues (fig. 5 b).

Researchers have used an aptamer peper that binds to and activates the fluorescent dye HBC (peppers can be inserted in both the uippm cell HES7 and MESP2 loci to image living cells for multiple RNA species) to enable us to monitor the oscillation of both RNAs. HES7 and MESP2 are expressed periodically in a dynamic manner in the anterior mesoderm of the somites. Indeed, HES7-GFP was expressed along the front end of UiSomitoid towards the back end, also supporting this feature (FIGS. 5c-5 e).

To more fully understand this gradient, we performed Geo-seq, and data analysis supported that UiSomitoid successfully established the anteroposterior axis (fig. 5f-5i, (a→p) referring to the anterior-to-posterior direction).

The results indicate that UiPSM can self-organize into a "tail of beak" structure (Somitoid) with a front-to-back axis and mimic the molecular clock of embryogenic body segments.

Those skilled in the art will further appreciate that the invention may be embodied in other specific forms without departing from the spirit or central attributes thereof. Since the foregoing description of the present disclosure provides exemplary embodiments thereof, it should be understood that other variations are within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the invention.

Reference to the literature

Beccari, L., moris, N., girgin, M., turner, D.A., baillie-Johnson, P., cossy, A.C., lutolf, M.P., duboule, D., and Arias, A.M. (2018), multi-axial self-organization properties of mouse embryonic stem cells into economides.Nature 562,272-276.

Henrique, d., abranshes, e., verrier, l., and store, k.g. (2015) Neuromesodermal progenitors and the making of the spinal cord.development 142,2864-2875.

Lee, t.j., jang, j., kang, s, bhang, s.h., jeong, g.j., shin, h., kim, d.w., and Kim, b.s. (2014) Mesenchymal stem cell-conditioned medium enhances osteogenic and chondrogenic differentiation of human embryonic stem cells and human induced pluripotent stem cells by mesodermal lineage reduction.tissue en Part a 20,1306-1313.

Li, R., zhong, C., yu, Y, liu, H, sakurai, M, yu, L, min, Z, shi, L, wei, Y, takahashi, Y, et al (2019), generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell cultures.cell 179,687-702e618.

Liao, b., bao, x., liu, l., feng, s, zovilis, a, liu, w, xue, y, cai, j, guo, x, qin, b., et al (2011), microRNA cluster 302-367enhances somatic cell reprogramming by accelerating a mesenchymal-to-ephelial transition.j Biol Chem 286,17359-17364.

Matthews, K.R.W., wagner, D.S., and Warmflash, A. (2021) Stem Cell-based models of embryos: the need for improved naming protocols.stem Cell Reports 16,1014-1020.

Moris, N., anlas, K., van den Brink, S.C., alemany, A., schroder, J., ghimire, S., balayo, T., van Oudenaarden, A., and Martinez Arias, A. (2020). An in vitro model of early anteroposterior organization during human development. Nature 582,410-415.

Nejadnik, h., diecke, s, lenkov, o.d., chapylin, f., donig, j., tong, x., derugin, n., chan, r.c., gaur, a., yang, f., et al (2015), improved approach for chondrogenic differentiation of human induced pluripotent stem cells, stem Cell Rev Rep 11,242-253.

Saito, s., and Suzuki, t. (2020). How do signaling and transcription factors regulate both axis elongation and Hox gene expression along the anteroposterior axisDev Growth Differ, 363-375.

Shelton, M., metz, J., liu, J., carpenedo, R.L., polymers, S.P., stanford, W.L., and Skerjanc, I.S. (2014) Derivation and expansion of PAX-positive muscle progenitors from human and mouse embryonic stem cells.

Takahashi, k, tanabe, k, ohnuki, m, narita, m, ichasaka, t, tomda, k, and Yamanaka, s. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131,861-872.Takahashi, k, and Yamanaka, s. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126,663-676.

van den Brink, s.c., baillie-Johnson, p., balayo, t., hadjanthoniasis, a.k., nowotschn, s., turner, d.a., and Martinez Arias, a. (2014), symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.development 141,4231-4242.

Wang, l., huang, w., su, h., xue, y, su, z, liao, b., wang, h., bao, x, qin, d., et al (2013), generation of integration-free neural progenitor cells from cells in human, nature Methods 10,84-89.

Yu, l., wei, y, duran, j, schmitz, d.a., sakurai, m., wang, l., wang, k., zhao, s., hon, g.c., and Wu, j (2021) Blastocyst-like structures generated from human pluripotent stem cells, nature 591,620-626.

Zhou, T., benda, C., dunzinger, S., huang, Y, ho, J.C., yang, J, wang, Y, zhang, Y, zhuang, Q, li, Y, et al (2012) Generation of human induced pluripotent stem cells from urine samples Nat Protoc 7,2080-2089.

Claims

1. A method of inducing urine cells to produce somite pre-mesodermal progenitor cells comprising the steps of:

(b) Inducing the epithelioid cells in basal medium supplemented with WNT agonist, DOT1L inhibitor, and one or more growth factors for a time sufficient to form granular colonies; and

(c) Extranodal mesodermal progenitor cells are obtained from the colonies.

2. The method of claim 1, wherein prior to step (b), the epithelial-like cells are pre-treated with one or more vectors capable of improving cell permeability for inducing or enhancing cell reprogramming.

3. The method of claim 2, wherein the vector expresses one or more of the following factors: oct4, sox2, SV40LT, klf4, miRNA 302, miRNA 303, miRNA 304, miRNA 305, miRNA 306, and miRNA 307.

4. A method according to claim 2 or 3, wherein after the pretreatment the epithelial-like cells are further cultured to restore their ability.

5. The method of any one of the preceding claims, wherein the urine cells are isolated from an upper urinary tract urine sample collected from one or more donor subjects.

6. The method of any one of the preceding claims, wherein in step (a), urine cells are cultured in REGM medium.

7. The method of any one of the preceding claims, wherein in step (b), the basal medium is DMEM, DMEM/F12 or Advanced DMEM/F2.

8. The method of any one of the preceding claims, wherein the growth factor is selected from FGF (e.g. bFGF), EGF, VEGF, PDGF, TGF- β, PD-ECGF, TNF, HGF, IGF (e.g. IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.

9. The method of any one of the preceding claims, wherein the growth factors are bFGF and EGF.

10. The method of any one of the preceding claims, wherein the WNT agonist is CHIR99021 and/or the DOT1L inhibitor is EPZ5676.

11. The method of claim 10, wherein the basal medium is Advanced DMEM/F12 supplemented with CHIR99021 in the concentration range of 2-4 μm (e.g., 3 μm), EPZ5676 in the concentration range of 4-6 μm (e.g., 5 μm), bFGF in the concentration range of 4-6ng/μl (e.g., 5ng/μl), and EGF in the concentration range of 4-6ng/μl (e.g., 5ng/μl).

12. The method of any one of the preceding claims, wherein the induction in step (b) is performed for 7-12 days, such as 9-10 days.

13. A somite anterior mesodermal progenitor cell obtained or obtainable by the method of any one of the preceding claims.

14. A method for culturing the pre-somite mesodermal progenitor cell of claim 13, comprising culturing the pre-somite mesodermal progenitor cell in a basal medium supplemented with a WNT agonist, a TGF- β inhibitor, and one or more growth factors.

15. The method of claim 14, wherein the basal medium is DMEM, DMEM/F12 or Advanced DMEM/F2.

16. The method of claim 14 or 15, wherein the WNT agonist is CHIR99021 and/or the TGF- β inhibitor is a8301.

17. The method of any one of claims 14-16, wherein the growth factor is selected from the group consisting of FGF (e.g., bFGF), EGF, VEGF, PDGF, TGF- β, PD-ECGF, TNF, HGF, IGF (e.g., IGF 1), BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.

18. The method of claim 17, wherein the selected growth factors are bFGF and EGF.

19. The method of claim 18, wherein the defined medium is Advanced DMEM/F12 supplemented with CHIR99021 in the concentration range of 2-4 μm (e.g. 3 μm), a8301 in the concentration range of 4-6 μm (e.g. 5 μm), bFGF in the concentration range of 4-6ng/μl (e.g. 5ng/μl) and EGF in the concentration range of 4-6ng/μl (e.g. 5ng/μl).

20. The method of any one of claims 14-19, further comprising passaging the cultured somite pre-mesodermal progenitor cells.

21. A somite anterior mesodermal progenitor cell obtained or obtainable by the method of any one of claims 14-20.

(b) Inducing the cells in basal medium supplemented with KSR, ITS, NEAA, β -ME, IGF-1, HGF, CHIR99021, VC, dex and SB 431542; and

(c) The cells were induced in basal medium supplemented with KSR, horse serum, NEAA, beta-ME, IGF-1 and HGF.

23. The method of claim 22, wherein in step (b), the basal medium is supplemented with 15% ksr, 1% its, 1% neaa, 0.1 μΜ β -ME, 4ng/ml IGF-1, 10ng/ml HGF, 3 μmchir99021, 50ng/ml VC, 0.5ng/ml Dex, and 2nm SB431542.

24. The method of claim 22 or 23, wherein in step (c) the basal medium is supplemented with 15% ksr, 2% horse serum, 1% neaa, 0.1 μΜ β -ME, 4ng/ml IGF-1 and 10ng/ml HGF.

25. The method of any one of claims 22-24, wherein in step (b) the induction is performed for a period of time in the range of 12-18 days, such as 15 days.

26. The method of any one of claims 22-25, wherein in step (c), the inducing is performed until skeletal muscle fiber bundles are fully present.

27. A method for differentiating somite anterior mesodermal progenitor cells into osteoblasts, comprising:

(b) The cells were induced in basal medium supplemented with FBS, VC, beta-glycerophosphate and 1-thioglycerol.

28. The method of claim 27, wherein in step (b), the basal medium is supplemented with 10% fbs, 50ng/ml VC, 100nM β -glycerophosphate and 1 μΜ 1-thioglycerol.

(b) The cells were induced in basal medium supplemented with FBS, ITS, sodium pyruvate, VC, beta-glycerophosphate, TGF-beta 3 and BMP2.

30. The method of claim 29, wherein in step (b), the basal medium is supplemented with 10% fbs, 1% its, 1% sodium pyruvate, 50ng/ml VC, 0.1nM β -glycerophosphate, 4ng/ml TGF- β3, and 20ng/ml BMP2.

31. The method of any one of claims 27-30, wherein the induction is performed for 15 days.

32. The method of any one of claims 22-31, wherein the basal medium is DMEM/F12 or DMEM.

33. The method of any one of claims 22-32, wherein the medium is exchanged every 2-3 days during induction.

34. A method for producing a somite structure, comprising:

(a) Inoculating the somite pre-mesodermal progenitor cells of claim 13 or 21 and culturing for a time sufficient to form a compact spherical cell aggregate;

(b) Extending the cell aggregates in basal medium supplemented with WNT agonist and NODAL inhibitor; and

(c) The extended structure is separated from the plate.

35. The method of claim 34, wherein in step (a), the culturing is performed in a medium supplemented with WNT agonist, TGF- β inhibitor, and one or more growth factors.

36. The method of claim 35, wherein the medium is Advanced DMEM/F12 supplemented with CHIR99021, a8301, bFGF and EGF.

37. The method of claim 36, wherein the medium is Advanced DMEM/F12 supplemented with CHIR99021 in the concentration range of 2-4 μm (e.g., 3 μm), a8301 in the concentration range of 4-6 μm (e.g., 5 μm), bFGF in the concentration range of 4-6ng/μl (e.g., 5ng/μl), and EGF in the concentration range of 4-6ng/μl (e.g., 5ng/μl).

38. The method of claim 34, wherein in step (b), the basal medium is Advanced DMEM/F12 supplemented with CHIR99021 and SB 431542.

39. The method of claim 38, wherein CHIR99021 is in the concentration range of 2-4 μm (e.g. 3 μm) and SB431542 is in the concentration range of 5-15 μm (e.g. 10 μm).

40. The method of any one of claims 34-39, wherein said tight spheroid cell aggregates are formed after about 48 hours of culture.

41. The method of any one of claims 34-40, wherein the cell aggregate is extended for a period of 7-12 days.

42. The method of any of claims 34-41, wherein the number of cells used for seeding is about 200-1000 cells, such as 300 cells, 400 cells, 500 cells, 600 cells, 700 cells, 800 cells, 900 cells and 1000 cells, preferably 400 cells.

43. A somite structure produced by the method of any one of claims 34-42.

44. The somite structure of claim 43, wherein said structure mimics an extended embryo tail bud and establishes an antero-posterior axis.

45. A composition comprising the somite pre-mesodermal progenitor cell of claim 13 or 21 or the somite structure of claim 44.

46. The use of the somite structure of claim 44 in modeling human disease of the mesodermal lineage.

47. The use of the somite structure of claim 44 in the screening of drugs including small molecule, protein and antibody based therapeutics.

48. Use of the somite pre-mesodermal progenitor cell of claim 13 or 21 or the composition of claim 45 for treating a disease or disorder in a subject in need of muscle regeneration, cartilage regeneration, spinal cord regeneration or related regeneration.

49. A method for treating a disease or disorder in a subject in need of muscle regeneration, cartilage regeneration or spinal cord regeneration, comprising administering (e.g., implanting) the pre-somite mesodermal progenitor cell of claim 13 or 21 or the somite structure of claim 45 to the subject.

a basal medium which is Advanced DMEM/F12;

combinations of agents for inducing urine cells, including CHIR99021, EPZ5676, bFGF and EGF, or CHIR9901, a8301, bFGF and EGF; and

51. A kit for differentiating the somite pre-mesodermal progenitor cells of claim 13 or 21, comprising:

a basal medium which is DMEM/F12 or DMEM; and

a combination of agents selected from the group consisting of:

(a) KSR, ITS, NEAA, beta-ME, IGF-1, HGF, CHIR99021, VC, dex and SB431542;

(b) KSR, horse serum, NEAA, beta-ME, IGF-1 and HGF;

(c) FBS, VC, beta-glycerophosphate and 1-thioglycerol; and

(d) FBS, ITS, sodium pyruvate, VC, beta-glycerophosphate, TGF-beta 3 and BMP2.