WO2023133726A1 - Cellules progénitrices du mésoderme présomitique induit, issues de l'urine humaine, et leurs utilisations - Google Patents

Cellules progénitrices du mésoderme présomitique induit, issues de l'urine humaine, et leurs utilisations Download PDF

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WO2023133726A1
WO2023133726A1 PCT/CN2022/071579 CN2022071579W WO2023133726A1 WO 2023133726 A1 WO2023133726 A1 WO 2023133726A1 CN 2022071579 W CN2022071579 W CN 2022071579W WO 2023133726 A1 WO2023133726 A1 WO 2023133726A1
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cells
presomitic
basal medium
progenitor cells
supplemented
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PCT/CN2022/071579
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Duanqing Pei
Yue QIN
Shangtao CAO
Xingnan HUANG
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Westlake University
Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences
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Definitions

  • the present application relates to human urine-derived induced presomitic mesoderm progenitor (UiPSM) cells, a somitoid structure formed by UiPSM cells, methods of producing the UiPSM cells and the somitoid structure, as well as uses thereof.
  • UiPSM urine-derived induced presomitic mesoderm progenitor
  • iPSCs has the potential to differentiate into germ layer, but the risk of tumorigenicity limits the application, hence, cells induced from iPSC also potentially own risk of tumorigenesis for the induction efficiency cannot reach 100%.
  • fibroblast For human somatic cell, a variety of cells including fibroblast, blood cells, amniotic cells, skin epithelial stem cells, proximal tubule (HK2) cell line, pericyte-derived cells, endothelial cells, pancreatic exocrine cells serve as sources of reprogramming induction initiation, while the acquisition of these cells could damage the subjects to some extent.
  • fibroblast For human somatic cell, a variety of cells including fibroblast, blood cells, amniotic cells, skin epithelial stem cells, proximal tubule (HK2) cell line, pericyte-derived cells, endothelial cells, pancreatic exocrine cells serve as sources of reprogramming induction initiation, while the acquisition of these cells could damage the subjects to some extent.
  • HK2 proximal tubule
  • pericyte-derived cells For human somatic cell, a variety of cells including fibroblast, blood cells, amniotic cells, skin epithelial stem cells,
  • non-invasively acquired urine cells could be reprogrammed into induced 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) , which suggest that urine cells as an initiative cell own potential to induce differentiation and certain advantages in clinical application.
  • iPSCs induced pluripotent stem cells
  • NPCs neural progenitor cells
  • Vertebrate embryos display a highly conserved characteristic spatial patterning of tissues.
  • the most compelling evidence for dual-fated neuromesodermal progenitor (NMP) cells comes from tailbud cell population with self-renewing properties contributes to both the spinal cord and paraxial mesoderm (Henrique et al., 2015) .
  • Presomitic mesodermal (PSM) cell is an axial stem cell derived from the caudal lateral epiblast (CLE) and could form somites along the anteroposterior axis, which determine axis extension during embryonic development (Henrique et al., 2015; Saito and Suzuki, 2020) .
  • the paraxial mesoderm (also called presomitic mesoderm) cell could develop into skeletal sclerotome, myotome and dermatome, which further differentiate into musculoskeletal system, while it’s hard to acquire self-renewing PSM cells in human embryo owing to the ethical and technical restrictions. Therefore, it’s important to build self-renewing PSM progenitor cells in vitro for somitogenesis research, while it’s hard to acquire self-renewing PSM cells in human embryo owing to the ethical and technical restrictions.
  • ES cells can differentiate into PSM and generate a three-dimensional ‘gastruloids’ model after aggregation in a defined conditional medium (Beccari et al., 2018; Moris et al., 2020) , it is unsufficient to imitate somitogenesis process owing to the occurrence of gastrulation and not a good model for accurate disease.
  • This result is mainly attributed to the fact that the cells of initiative aggregation lack PSM properties, such as self-renewing and multipotential differentiation of presomitic mesodermal lineage cell (muscle cell and bone cell) . It’s crucial to generate self-renewing PSM cells to build a pure somitoid to mimic somitogenesis on early embryonic development.
  • the present disclosure shows that urine cell could be reprogramed into presomitic mesoderm progenitor cells, which expand steadily and differentiate into mesodermal lineage cells.
  • the presomitic mesoderm progenitor cells can also self-organize into a somitoid structure to mimic somitogenesis.
  • the generated presomitic mesoderm progenitor cells are designated herein as urine-derived induced presomitic mesodermal progenitor (abbreviated as UiPSM) cells.
  • UiPSM cells show high expression of presomitic mesoderm related genes and are characterized with a presomitic mesodermal transcription characteristic expression profile. Importantly, UiPSM cells drop out pluripotent state and have reduced risk of tumor formation, while retaining the potential to differentiate into presomitic lineage cells.
  • the UiPSM cells can self-organize in vitro to generate a UiSomitoid structure, which is a UiPSM self-organized ‘rostral-caudal’ structure similar to somite.
  • the UiSomitoid structure imitates the establishment of similar tissue in somite formation during early embryonic development and mainly involved in stem cell-based models of embryos (SCMEs) .
  • SCMEs stem cell-based models of embryos
  • the UiSomitoid structure could better simulate the anteroposterior axis and the establishment of molecular clocks of embryonic somitogenesis.
  • a method of inducing urine cells to obtain presomitic mesoderm progenitor cells (UiPSM cells) .
  • the method may comprise the following steps:
  • the urine cells are obtained from upper urinary tract urine samples collected from one or more subject (s) .
  • the urine cells are cultured with REGM medium, and mainly epithelial-like and mesenchymal cell types are enriched during the cultivation.
  • the epithelial-like cells are transformed by electroporation of one or more vectors, to make the cells susceptible to subsequent induction.
  • the vectors used may be pEP4E02SET2K and pCEP4-miR-302-367.
  • the state of urinary cells restored in 2 days after the electroporation.
  • the growth factor (s) used is/are selected from FGF (e.g. bFGF) , EGF, VEGF, PDGF, TGF- ⁇ , PD-ECGF, TNF, HGF, IGF (e.g. IGF 1) , BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.
  • the basal medium used for inducing the epithelial-like cells are Advanced DMEM/F12.
  • the WNT agonist is CHIR99021.
  • the DOT1L inhibitor is EPZ5676.
  • the induction is performed in a time period of about 7 to 12 days.
  • presomitic mesodermal progenitor cells obtained by the method as described herein.
  • the UiPSM cells have the capability to differentiate into presomitic mesodermal lineage cells, such as skeletal muscle cells, osteoblasts, chondrocytes and chondroblasts.
  • a somitoid structure comprising the presomitic mesoderm progenitor cells derived from urine cells as described above.
  • the somitoid structure are produced by expanding and differentiating the presomitic mesoderm progenitor cells in vitro.
  • the somitoid structure is self-organized ‘rostral-caudal’ structure.
  • provided he somitoid structure rein is a method of producing the somitoid structure as described above, comprising:
  • the WNT agonist is CHIR99021.
  • the NODAL inhibitor is SB431542.
  • Urin-derived presomitic mesoderm progenitor cells (UiPSM cells) in the induced differentiation of skeletal muscle cell, osteoblast and chondrocyte.
  • a reprogramming system for presomitic mesoderm progenitor cells induced from urine cells UiPSM cells.
  • a method of inducing urine cells to generate presomitic mesoderm progenitor cells comprising the following steps:
  • step (b) the epithelial-like cells are pretreated with a vector (s) that could improve permeability of the cells for induction or enhance cell reprogramming.
  • step (a) the urine cells are cultured in REGM medium.
  • step (b) the basal medium is DMEM, DMEM/F12 or Advanced DMEM/F12.
  • the growth factor (s) is/are selected from FGF (e.g. bFGF) , EGF, VEGF, PDGF, TGF- ⁇ , PD-ECGF, TNF, HGF, IGF (e.g. IGF1) , BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.
  • the basal medium is Advanced DMEM/F12 which is supplemented with CHIR99021 at a concentration range of about 2-4 ⁇ M (e.g. 3 ⁇ M, 2.5-3 ⁇ M, 3-3.5 ⁇ M) , EPZ5676 at a concentration range of about 4-6 ⁇ M (e.g. 5 ⁇ M, 4.5-5 ⁇ M, 5-5.5 ⁇ M) , bFGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5 ng/ ⁇ l, 5-5.5ng/ ⁇ l) and EGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5 ng/ ⁇ l, 5-5.5ng/ ⁇ l) .
  • step (b) is performed for a period of about 7 to 12 days, such as about 7 days, about 8 days, about 9 days, about 10 days, about 11 days and about 12 days.
  • presomitic mesodermal progenitor cells obtained or obtainable by the method of any of the preceding embodiments.
  • a method for culturing the presomitic mesodermal progenitor cells of embodiment 13, comprising culturing the presomitic mesodermal progenitor cells in a basal medium supplemented with a WNT agonist, a TGF- ⁇ inhibitor and one or more growth factor (s) .
  • the growth factor (s) is/are selected from FGF (e.g. bFGF) , EGF, VEGF, PDGF, TGF- ⁇ , PD-ECGF, TNF, HGF, IGF (e.g. IGF1) , BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.
  • the defined medium is Advanced DMEM/F12 supplemented with CHIR99021 at a concentration range of about 2-4 ⁇ M (e.g. 3 ⁇ M, 2.5-3 ⁇ M, 3-3.5 ⁇ M) , A8301 at a concentration range of about 4-6 ⁇ M (e.g. 5 ⁇ M, 4.5-5 ⁇ M, 5-5.5 ⁇ M) , bFGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5 ng/ ⁇ l, 5-5.5ng/ ⁇ l) and EGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5 ng/ ⁇ l, 5-5.5ng/ ⁇ l) .
  • A8301 at a concentration range of about 4-6 ⁇ M (e.g. 5 ⁇ M, 4.5-5 ⁇ M, 5-5.5 ⁇ M)
  • bFGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5
  • a method for differentiating presomitic mesodermal progenitor cells into skeletal muscle cells comprising:
  • step (b) the basal medium is supplemented with about 15%KSR, about 1%ITS, about 1%NEAA, about 0.1 ⁇ M ⁇ -ME, about 4 ng/ml IGF-1, about 10ng/ml HGF, about 3 ⁇ M CHIR99021, about 50ng/ml VC, about 0.5ng/ml Dex and about 2nM SB431542.
  • step (c) the basal medium is supplemented with about 15%KSR, about 2%Horse serum, about 1%NEAA, about 0.1 ⁇ M ⁇ -ME, about 4 ng/ml IGF-1, and about 10 ng/ml HGF.
  • step (b) the induction is performed for a time period in the range of 12-18 days, such as 15 days, 12 days, 13 days, 14 days, 16 days, 17 days and 18 days.
  • step (c) the induction is performed until skeletal muscle fiber bundles are fully present.
  • a method for differentiating presomitic mesodermal progenitor cells into osteoblasts comprising:
  • step (b) the basal medium is supplemented with about 10%FBS, about 50ng/ml VC, about 100 nM ⁇ -Glycerophosphate and about 1 ⁇ M 1-Thioglycerol.
  • a method for differentiating presomitic mesodermal progenitor cells into chondrocytes comprising:
  • step (b) the basal medium is supplemented with about 10%FBS, about 1%ITS, about 1%Sodium Pyruvate, about 50ng/ml VC, about 0.1 nM ⁇ -Glycerophosphate, about 4 ng/mL TGF- ⁇ 3 and about 20ng/ml BMP2.
  • a method for producing a somitoid structure comprising:
  • step (a) the culture is performed in a medium supplemented with a WNT agonist, a TGF- ⁇ inhibitor and one or more growth factor (s) .
  • step (b) the basal medium is Advanced DMEM/F12 which is supplemented with CHIR99021 and SB431542.
  • CHIR99021 is at a concentration range of about 2-4 ⁇ M (e.g. 3 ⁇ M, 2.5-3 ⁇ M, 3-3.5 ⁇ M)
  • SB431542 at a concentration range of about 5-15 ⁇ M (e.g. 10 ⁇ M, 5-10 ⁇ M, 10-15 ⁇ M) .
  • any of embodiments 34-41, wherein the number of cells 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 about 400 cells.
  • a composition comprising the presomitic mesoderm progenitor cells of embodiment 13 or 21 or the somitoid structure of embodiment 44.
  • a method for treating a disease or disorder in a subject in need of muscle regeneration, cartilage regeneration, spinal cord regeneration comprising administering (e.g. implanting) the presomitic mesoderm progenitor cells of embodiment 13 or 21 or the somitoid structure of embodiment 45 to a subject.
  • a kit for reprogramming urine cells to presomitic mesoderm progenitor cells comprising:
  • a basal medium which is Advanced DMEM/F12;
  • reagents for inducing the urine cells including CHIR99021, EPZ5676, bFGF, and EGF, or CHIR99021, A8301, bFGF, and EGF; and
  • tools for collecting urine cells or picking epithelial-like cells optionally, tools for collecting urine cells or picking epithelial-like cells.
  • a basal medium which is DMEM/F12 or DMEM
  • FIG. 1 shows the generation of UiPSM.
  • Figure 1a shows a schematic representation of the vertebrate embryonic central nervous system indicating cell populations that give rise to the CNS.
  • the posterior spinal cord arises from neuromesodermal progenitors (NMps; red/green) , which are located in the anterior primitive streak (PS; brown) and in the adjacent caudal lateral epiblast (CLE; light grey) .
  • NMps have given rise to new neural progenitors (Np; green) , which contribute to the CLE (light grey) and then the preneural tube (PNT; dark grey) , and to new mesoderm progenitors (Mp; red) , which contribute to presomitic mesoderm (PSM; brown) , then give rise to somites.
  • Figure 1b shows a schematic overview of UiPSM reprogramming from UC.
  • UC human derived urine cells
  • UiPSM presomitic mesoderm induced from human derived urine cells. Representative images show the cellular morphologic change during the whole UiPSM reprogramming. Scale bars, 100 ⁇ m.
  • Figure 1c shows immunofluorescence of T (green) co-stained with MIXL1 (red) (upper panel) , TBX6 (green) co-stained with CDX2 (red) (middle panel) , SOX2 (green) co-stained with SALL4 (red) (bottom panel) of the UiPSM at day 9.
  • the scale bar represents 200 ⁇ m.
  • Figure 1d shows the expression of PSM-specific genes T, MIXL1, TBX6 and CDX2 during the whole reprogramming process.
  • Figure 1e shows representative flow cytometric inducible effective evaluation of T protein expression during the whole UiPSM reprogramming.
  • HPS human induce primitive streak
  • Figure 1f shows t-SNE projection of all 32798 individual cells at day0, day3, day6, day9 during the whole reprogramming process, cells were colored by indicated time points.
  • Figure 1g shows typical marker expression plotted on t-SNE projection.
  • PSM markers CDX1, CDX2, DLL3, HES7, MIXL1, LEF1, TBX6, WNT5B, HOXB1, FGF8, T, MSX1 highly expressed at day9, kidney epithelium markers (PAX8, EPCAM, AHI1) enriched at day0, slightly expressed at day3, protein synthesis related genes (CD44, FN1, ITGA3) mainly expressed during the process including day3 and day6.
  • Figure 1h shows enriched gene ontology analyses of each cluster. P value was less than 0.05.
  • FIG. 1 shows that UiPSM colonies expand rapidly.
  • Figure 2a shows representative images on the generation of UiPSM treated in DM in different passages. Scale bars, 100 ⁇ m.
  • DM UiPSM maintain medium.
  • Figure 2b shows immunofluorescence of T (green) co-stained with MIXL1 (red) on the left, TBX6 (green) co-stained CDX2 (red) on the right in different passages of UiPSMs.
  • the scale bar represents 200 ⁇ m.
  • Figure 2c shows representative gene expression of presomitic mesoderm (T, MIXL1, TBX6, CDX2) of the UiPSM colonies derived from three individuals.
  • Figure 2d shows growth curve of UCs and UiPSMs.
  • Figure 2e shows correlation analysis showing the similarity among UiPSM and difference between UC and UiPSM.
  • UC-1#and UC-2# refer to UCs obtained from Donor 1 and 2
  • UiPSM1 and UiPSM2 refer to UiPSM differentiated from UC-1#1 and UC-2#, respectively.
  • Figure 2f shows enriched GO terms and p values of the UiPSM colonies on day 9 and p18.
  • Figure 3 shows UiPSM colonies maintain mesodermal features in vivo.
  • Figure 3a shows representative images showing morphological size differences of UiPSM and UiPSC clone teratomas on one month.
  • Figure 3b shows representative images of H&E staining of UiPSM and UiPSC clone teratomas derived from Urine cells.
  • Figure 3c shows t-SNE projection of all 12456 individual cells of UiPSM and UiPSC clone tetratomas derived from urine cells.
  • Figure 3d shows t-SNE projection colored by clusters identified by Louvain algorithm.
  • Figure 3e shows t-SNE plot of subpopulations of M1 cluster (bottom) .
  • Figure 3f shows gene ontology analyses for each cluster in Fig. 3d. P value ⁇ 0.05.
  • Figure 3g shows enriched gene ontology analyses for each subcluster in Fig. 3e. P value ⁇ 0.05.
  • Figure 4 shows differentiation of UiPSM into mature mesodermal cell types in vitro.
  • Figure 4a shows a schematic overview of stepwise differentiation of skeletal muscle cells (SKM) from UiPSM.
  • the bottom panel shows the morphological changes from UiPSM cells to skeletal muscle filaments. Scale bars, 100 ⁇ m.
  • Figure 4c shows immunofluorescence of MYOD, MF20, DESMIN, LAMIN during the differentiation of skeletal muscle cell from UiPSM at day 60. Scale bars, 100 ⁇ m. The bottom values represent the percentages of positive cells statistically.
  • Figure 4d shows enriched GO terms of the UiPSM and differentiated skeletal muscle cells at day 30 and day 60.
  • Figure 4e shows a schematic overview of chondroblasts differentiation from UiPSM Alcin blue staining of chondroblasts at day 15. Scale bars, 100 ⁇ m.
  • Figure 4f shows representative gene expression of chondroblast (ACAN, COL9A1, SOX9, COL2A1) during the differentiation process.
  • Figure 4g shows representative gene ontology enrichment terms in UiPSM and chondroblast based on up-regulated genes in each sample.
  • Figure 4h shows schematic overview of osteoblasts differentiation from UiPSM. Alizarin red staining of osteoblasts at day 15. Scale bars, 100 ⁇ m.
  • Figure 4i shows representative gene expression of osteoblast (OCN, SP7, BMP2, RUNX2) during the differentiation process.
  • Figure 4j shows representative gene ontology enrichment terms in UiPSM and osteoblast based on up-regulated genes in each sample.
  • Figure 5 shows UiPSM slef-organize into UiSomitoid with anteroposterior axis and established moleculr clocks of embryonic somitogenesis.
  • Figure 5a shows a schematic overview of the generation of UiSomitoids from UiPSM treated within 9 days after 2-day aggregation.
  • the bottom panel shows the enlongation of UiSomitoids. Scale bars, 200 ⁇ m.
  • Figure 5b shows immunofluorescence of SOX2 (green) co-stained with T (red) or CDX2 (red) within UiSomitoids at day 9.
  • the scale bar represents 200 ⁇ m.
  • Figure 5c shows the fluorescence signal of 8pepper was measured with Incucyte S3 Live-Cell Analysis System, converted into a digital signal via ImageJ, then normalized to maximum oscillation peak. Finally drawing oscillation trend line using ‘Sine wave with noezero baseline’ on prism 8. Period was calculated as average peak-to-peak (n>10) .
  • Figure 5d shows statistical period variation of HES7 and MESP2 reporter via recording changes in parameter ‘wavelength’ in ‘e’ .
  • Figure 5e shows statistical amplitude variation of HES7 and MESP2 reporter via recording changes in parameter ‘amplitude’ in ‘e’ , which was normalized to baseline.
  • Figure 5f illustrates somitogenesis in mammal embryos. Dark yellow, FGT/WNT signaling concentrated distribution region; light yellow, RA/BMP signaling intensive distribution area; green, dynamic expression of HES7. The bottom images show the enlongation of the UiSomitoids along A-P axis (green HES7-GFP) . Scale bars, 400 ⁇ m.
  • Figure 5g shows bar charts of the anteroposterior organization of tailbud-associated gene expressions in UiSomitoid, as T, SOX2 and CDX2.
  • Figure 5h shows transcriptomic analysis of Geo-seq, 1500 highly variable genes were selected by calculating variances along the anteroposterior axis of UiSomitoid. Selected genes were classified into 6 groups by fuzzy clustering method.
  • Figure 5i shows gene trends along the anteroposterior axis for selected gene clusters and corresponding enriched GO terms in UiSomitoid.
  • 1%ITS or 1%Sodium Pyruvate refers to a weight percentage of ITS or Sodium Pyruvate in the prepared medium.
  • mesoderm is the area of mesoderm in the neurulating embryo that flanks and forms simultaneously with the neural tube. The cells of this region give rise to somites, blocks of tissue running along both sides of the neural tube, which form muscle and the tissues of the back, including connective tissue and the dermis.
  • presomitic mesoderm are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. It has been found that WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. The ability to generate PSM cells with high efficiency in vitro can promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm.
  • mesoderm progenitor cells are a population of undifferentiated progenitor cells that originate in the early gastrula embryo. By the end of gastrulation, they have moved to the very posterior end of the embryo in a region called the tailbud. Throughout gastrulation and somitogenesis, the mesoderm progenitor cells continuously contribute cells to the presomitic mesoderm, producing a species-specific number of segments using a complex clock and wavefront mechanism to segment the presomitic mesoderm (for review, see Tam et al. Curr Top Dev Biol. 2000, 47: 1-32; Holley and Takeda, Semin Cell Dev Biol. 2002 Dec, 13 (6) : 481-8; Dubrulle and Pourquie, Development. 2004 Dec, 131 (23) : 5783-93) .
  • reprogramming refers to a process that alters or reverses the differentiation status of a somatic cell (e.g. urine cell) that is either partially or terminally differentiated.
  • Reprogramming of a somatic cell may be a partial or complete reversion of the differentiation status of the somatic cell.
  • reprogramming is complete when a somatic cell is reprogrammed into an induced pluripotent stem cell.
  • reprogramming is partial, such as reversion into any less differentiated state.
  • reverting a terminally differentiated cell into a cell of a less differentiated state such as a multipotent cell, a progenitor cell (such as presomitic mesoderm progenitor) .
  • the reprogramming process may be caused by inducing the somatic cells with a set of small molecule compounds (such as WNT agonist, DOT1L inhibitor and growth factors) , or by expression of transcription factors and micro-RNAs in the somatic cells (such as POU5F1, KLF4, SOX2, c-MYC and MIR302-367 cluster) .
  • reprogramming urine cells to presomitic mesodermal progenitor cells can be achieved via induction by a specific combination of factors and compounds.
  • GO Gene ontology
  • Gene ontology can be obtained from the Gene Ontology Consortium and usually consist of a set of classes (or terms or concepts) with relations that operate between them. Gene ontology can be applied to describe the roles of genes and gene products in all organisms. Gene Ontology can characterize the relationships between genes and the keywords assigned for each gene. Gene ontology terms for e.g. biological process, cellular component, and molecular function can be found at http: //www. geneontology. org. GO analysis can be done, for example, using the EASE software.
  • the term “passage” designates the step of detaching the cells from their support (by means of an enzyme or cocktail of enzymes) and diluting the cells in the culture medium prior to their seeding on a new support for growth.
  • p9 herein refers to cells that have been passaged 9 times.
  • the cells for use as a starting material in the reprogramming or induction herein are human derived urine cells.
  • the present disclosure shows that urine cells are an ideal source for functional lineage-specific cells in terms of favorable gene profile and inherent multipotent potential. Instead of generating urine stem cells from urine cells, generating functional lineage-specific progenitor cells can drop out pluripotent state and have reduced risk of tumor formation, thus have a prospective future for application in clinical therapy.
  • reprogramming the human urine cells successfully generates presomitic mesoderm (PSM) progenitor cells, which may further differentiate into presomitic mesodermal lineage cells (e.g. muscle cells and bone cells) .
  • PSM presomitic mesoderm
  • Urine samples may be collected from subjects in a simple and non-invasive manner.
  • the urine cells may be isolated from urine samples using a cost-effective and simple method of isolation.
  • Non-invasiveness and easy isolation are the main advantages of urine-derived cells compared with all other donor-related samples.
  • Adipose-derived stem cells, hair cells, fibroblasts, amniotic cells, skin epithelial stem cells, proximal tubule (HK2) cell line, pericyte-derived cells, endothelial cells, pancreatic exocrine cells and mesenchymal stromal cells require liposuction or invasive methods for sample isolation; Amniotic and umbilical cord cells are neither easily accessible nor suitable for auto-transplantation.
  • urine cells can be isolated by simple centrifugation of the samples to sediment the cells and by culturing and expanding them in cell growth medium (such as REGM medium) .
  • the cell medium may be selected to promote the growth of certain type of urine cells.
  • the growth medium used for expanding the urine cells promote the growth and viability of epithelioid (epithelial-like) cells, thus the epithelioid (epithelial-like) and mesenchymal cell types may be enriched during expansion.
  • Epithelial-like cells may be obtained by picking with a tool (e.g. a long glass pasteur straw) when collecting the adherent urine cells grown into larger lumps on the plate, or rapidly digesting the epithelial-like cells with 0.25%trypsin-EDTA on a new plate, this method is due to epithelial-like cells can be more easily digested than mesenchymal cells.
  • a tool e.g. a long glass pasteur straw
  • the epithelial-like cells enriched in urine cell cultivation are picked for further induction.
  • the epithelioid cells are pretreated via electroporating with vectors (e.g. pEP4EO2SET2K and pCEP4-miR-302-367) to make the cells susceptible to induction.
  • vectors e.g. pEP4EO2SET2K and pCEP4-miR-302-367
  • pEP4EO2SET2K is a non-integrating episomal vector encoding OCT4, SOX2, SV40LT, KLF4, and
  • pCEP4-miR-302-367 is a non-integrating episomal vector encoding miR302-367 cluster, thus can be used for promoting cell permeability.
  • the epithelial-like cells may be incubated in a basal medium supplemented with a selected group of compounds and molecules.
  • the urine derived epithelial-like cells are induced in a basal medium supplemented with WNT agonist CHIR99021, DOT1 inhibitor EPZ5676, and one or more growth factor (s) selected from FGF (e.g. bFGF) , EGF, VEGF, PDGF, TGF- ⁇ , PD-ECGF, TNF, HGF, IGF (e.g. IGF 1) , BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.
  • the basal medium may be supplemented with bFGF and EGF.
  • the basal medium is Advanced DMEM/F12.
  • the basal medium is supplemented with CHIR99021 at a concentration range of 2-4 ⁇ M (e.g. 3 ⁇ M) , EPZ5676 at a concentration range of 4-6 ⁇ M (e.g. 5 ⁇ M) , bFGF at a concentration range of 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l) and EGF at a concentration range of 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l) .
  • the induction may last for about 7 to 12 days, until epithelial-like cell tightly joined together to form clones are observed.
  • the morphology, biological and molecular changes taking place on the induced cells at different time points can be detected by a variety of methods and assays.
  • the obtained granulated colonies are not similar to human primer and cells or NPCs in morphology.
  • These reprogrammed or induced cells are named as UiPSM cells (Urine derived presomitic mesodermal progenitor cell) .
  • markers have been known to be related to presomitic mesoderm cells, including T (also known as Brachyury and TBXT) , MIXL1, TBX6 and CDX2.
  • T also known as Brachyury and TBXT
  • MIXL1 RNA-seq sequencing
  • FACS FACS
  • pluripotential markers such as POU5F1 and NANOG
  • endodermal marker SOX17 and FOXA2
  • ectodermal marker such as SOX1 and PAX6 hardly expressed. Therefore, pluripotency is not required during the reprogramming process, the generated presomitic mesoderm progenitor cells have no indicated endodermal and ectodermal features, reducing the risk of tumor formation in vivo.
  • Tables A and B describe the genes highly expressed and little expressed in UiPSM cells, respectively.
  • the reprogrammed UiPSM cells are further characterized in that they can stably and rapidly proliferate in vitro.
  • the UiPSM cells are cultured or passaged in a defined medium that could maintain the stable expansion of UiPSM.
  • the defined medium comprises WNT agonist CHIR99021, TGF- ⁇ inhibitor A8301, and one or more growth factor (s) selected from FGF (e.g. bFGF) , EGF, VEGF, PDGF, TGF- ⁇ , PD-ECGF, TNF, HGF, IGF (e.g. IGF 1) , BMP, erythropoietin, CSF, M-CSF, and fragments or variants thereof.
  • the selected growth factors may be bFGF and EGF.
  • the basal medium is Advanced DMEM/F12.
  • the defined medium comprises CHIR99021 at a concentration range of 2-4 ⁇ M (e.g. 3 ⁇ M) , A8301 at a concentration range of 4-6 ⁇ M (e.g. 5 ⁇ M) , bFGF at a concentration range of 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l) and EGF at a concentration range of 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l) .
  • a concentration range of 2-4 ⁇ M means an approximate range of 2-4 ⁇ M and includes any value and subrange contained in the range, such as 2 ⁇ M, 2.2 ⁇ M, 2.4 ⁇ M, 2.6 ⁇ M, 2.8 ⁇ M, 3 ⁇ M, 3.2 ⁇ M, 3.4 ⁇ M, 3.6 ⁇ M, 3.8 ⁇ M, 4 ⁇ M.
  • a concentration range of 4-6 ⁇ M means an approximate range of 4-6 ⁇ M and includes any value and subrange contained in the range, such as 4 ⁇ M, 4.2 ⁇ M, 4.4 ⁇ M, 4.6 ⁇ M, 4.8 ⁇ M, 5 ⁇ M, 5.2 ⁇ M, 5.4 ⁇ M, 5.6 ⁇ M, 5.8 ⁇ M, 6 ⁇ M;
  • a concentration range of 5-15 ⁇ M means an approximate range of 5-15 ⁇ M and includes any value and subrange contained in the range, such as 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M;
  • a concentration range of 4-6 ng/ ⁇ l means an approximate range of 4-6 ng/ ⁇ l and includes any value and subrange contained in the range, such as 4 ng/ ⁇ l, 4.2 ng/ ⁇ l, 4.4ng/ ⁇ l, 4.6ng/ ⁇ l, 4.8ng/ ⁇ l, 5ng/ ⁇
  • UiPSM cells maintain their characteristics as the first-generation of UiPSMs induced from urine cells.
  • qRT-PCR, Immunofluorescence and RNA-seq supported that continuously passaged UiPSMs continue to stably express presomitic markers T, MIXL1, TBX6 and CDX2.
  • the generated presomitic mesoderm progenitor cells have specific stemness for they have self-renewal abilities and specific differentiative capacities of presomitic mesoderm lineage cells in various selected optimal mediums.
  • the presomitic mesoderm progenitor cells (UiPSMs) cells can be successfully differentiated to osteoblast, chondroblast and skeletal muscle cells in vitro.
  • the UiPSM cells obtained by the methods of the disclosure may be cultured in vitro under differentiation conditions, to generate desired cells. There are various differentiation methods known in the art (see Zhou et al., Generation of human induced pluripotent stem cells from urine samples.
  • the UiPSM cells can be used for differentiating the UiPSM cells herein.
  • optimized differentiation protocols for different target cells may be used.
  • the UiPSM cells have the capability to be differentiated to muscle cells, osteoblasts, osteocytes and chondrocyte, respectively, under defined conditions.
  • Q-PCR, Immunofluorescence, FACS and bulk RNA-seq data supported the successful establishment of the above self-renewal and presomitic lineage specific differentiation system.
  • the skeletal muscle cells differentiated from the presomitic mesoderm progenitor cells have similar gene expression profile as the naturally existing skeletal muscle cells and participate in muscle regeneration.
  • a similar expression profile means that the relative expression profile of a marker in the UiPSM cells of the disclosure is similar to the relative expression profile of the published naturally existing presomitic mesoderm progenitor cell.
  • the osteoblast or osteocytes differentiated from the presomitic mesoderm progenitor cells have similar gene expression profile as the naturally existing osteoblast or osteocytes.
  • the chondrocytes differentiated from the presomitic mesoderm progenitor cells have similar gene expression profile as the naturally existing chondrocytes cells.
  • Urine-derived presomitic mesoderm progenitor cells self-organize to form UiSomitoid
  • the presomitic mesoderm progenitor cells have the ability to self-organize in vitro to form a specific somite-like structure, named as UiSomitoid herein.
  • UiPSM cells e.g. about 200-600, about 300-500, about 400 UiPSM cells
  • the process is preferably first performed in a defined medium to form compact spherical aggregates and then in a basal medium supplemented with WNT agonist and NODAL inhibitor to elongate the aggregates.
  • the defined medium is Advanced DMEM/F12 supplemented with CHIR99021 at a concentration range of about 2-4 ⁇ M (e.g. 3 ⁇ M, 2.5-3 ⁇ M, 3-3.5 ⁇ M) , A8301 at a concentration range of about 4-6 ⁇ M (e.g. 5 ⁇ M, 4.5-5 ⁇ M, 5-5.5 ⁇ M) , bFGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5 ng/ ⁇ l, 5-5.5ng/ ⁇ l) and EGF at a concentration range of about 4-6 ng/ ⁇ l (e.g. 5ng/ ⁇ l, 4.5-5 ng/ ⁇ l, 5-5.5ng/ ⁇ l) .
  • the basal medium for elongation is supplemented with WNT agonist CHIR99021 and NODAL inhibitor SB431542.
  • the basal medium is supplemented with WNT agonist CHIR99021 at a concentration range of 2-4 ⁇ M (e.g. 3 ⁇ M) and NODAL inhibitor SB431542 at a concentration range of 5-15 ⁇ M (e.g. 10 ⁇ M) .
  • the 3D structure is termed UiSomitoid, which display a ‘rostral’ cell-dense region and a polar extension towards a ‘caudal’ extremity, and resemble the elongating embryonic tailbud.
  • the UiSomitoid has a ‘rostral-caudal’ structure with anteroposterior axis and can be used to establish molecular clocks of embryonic somitogenesis.
  • optimization of the UiSomitoid generation procedure may take into consideration other factors, such as cell densities, inhibitors for BMP, Nodal signaling during aggregation and elongation steps, and so on.
  • the UiSomitoid structure provided herein displays polarized patterns of expression for SOX2/CDX2, SOX2/T, and CDX2/T in pairwise co-staining with antibodies, suggesting an anterior-posterior organization.
  • Geo-seq data analysis supported that UiSomitoid successfully establish anteroposterior axis.
  • UiPSM can self-organize into a ‘rostral-caudal’ structure (Somitoid) with anteroposterior axis and establish molecular clocks of embryonic somitogenesis.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • PSCs pluripotent stem cells
  • Mammalian ESCs and human ESCs were induced to develop gastruloids which are similar to a developing embryo at gastrulation stage (Beccari et al., 2018; Moris et al., 2020; van den Brink et al., 2014) .
  • embryoids allow theories and hypotheses to be tested and improved, can be easily manipulated, genetically manipulated or generated in diverse ways to improve the experimental design, and allow detailed observations of cellular events in real time when combined with fluorescent reporter constructs. These embryoids can also be produced in larger numbers to allow for statistical analysis, while it’s hard to do so on human embryos due to limited funding restrictions and ethical concerns (Matthews et al., 2021) .
  • NMP neuromesodermal progenitor
  • ES cells can differentiate into PSM, and generate three-dimensional ‘gastruloids’ models, it is complicated to study somitogenesis owing to gastrulation process.
  • iPSCs has the potential to differentiate into germ layer, while it has the risk of tumorigenicity limits the application, hence, cells induced from iPSC also potentially own risk of tumorigenesis for the induction efficiency cannot reach 100%.
  • fibroblast fibroblast
  • blood cells amniotic cells
  • skin epithelial stem cells proximal tubule (HK2) cell line
  • pericyte-derived cells pericyte-derived cells
  • endothelial cells pancreatic exocrine cells
  • pancreatic exocrine cells serve as sources of reprogramming induction initiation, while the acquisition of these cells could damage the subjects to some extent.
  • non-invasively acquired UCs could be reprogrammed into iPSCs and NPCs, which suggested that UC served as a good source for reprogramming.
  • the UiSomitoid structure are a self-organized and self-renewing structure, with features of somite development including the establishment of anteroposterior axis and molecular clocks of embryonic somitogenesis.
  • the UiSomitoid structure thus is a promising tool for studying somitogenesis.
  • the present invention provides a UiPSM reprogramming system from urine cells with high efficiency, the established UiPSM cells highly express presomitic mesoderm related genes including T, MIXL1, TBX6, CDX2 and built presomitic mesodermal transcription characteristic expression profile. Moreover, the generated UiPSM cells have no indicated pluripotential, endodermal and ectodermal features, and reduce the risk of tumor formation in vivo.
  • UiPSM colonies could expand steadily in vitro and have specific differentiation potential related presomitic mesodermal lineage in vivo or in vitro. Furthermore, UiPSM-derived skeletal muscle cells could survive and participate in muscle regeneration.
  • the stable expanded UiPSM colonies could self-organize into a ‘rostral-caudal’ structure (UiSomitoid) with anteroposterior axis and the establish molecular clocks of embryonic somitogenesis.
  • the inventors establish self-renewing UiPSM cells reprogrammed from urine cells, which can differentiate into different somite lineage cells and self-organize into a ‘rostral-caudal’ structure (UiSomitoid) with anteroposterior axis characteristic of somitogenesis in vitro.
  • the UiPSMs obtained or obtainable from the methods of the disclosure may advantageously be cultured in vitro under differentiation conditions to generate differentiated cells, especially presomitic mesodermal lineage cells, such as skeletal muscle cells, osteoblasts, chondrocytes and chondroblasts, muscle, cartilage, bone, dermal tissue, among others.
  • differentiated cells especially presomitic mesodermal lineage cells, such as skeletal muscle cells, osteoblasts, chondrocytes and chondroblasts, muscle, cartilage, bone, dermal tissue, among others.
  • the skilled person may use known protocols for differentiating stem cells, such as the protocols conventionally used for differentiating induced pluripotent stem cells, ES cells or mesenchymal stem cells into the desired cell lineages.
  • those protocols may be optimized based on specific needs.
  • UiPSM and UiSomitoid are major fields of application of UiPSM and UiSomitoid.
  • urine cells obtained from a subject may be cultured and subsequently reprogrammed into UiPSMs according to the methods of the present disclosure and differentiated into the suitable cell lineages for re-administration into the subject, for example the same subject as the cell donor (autologous treatment) .
  • regenerative medicine can be used to potentially cure any disease that results from malfunctioning, damaged or failing muscle or bone tissue by either regenerating the damaged tissues in vivo by direct in vivo implanting of a composition comprising UiPSMs or their derivatives comprising appropriate progenitors or cell lineages.
  • the reprogrammed UiPSMs may be useful for autologous regenerative therapy of a patient in need of regenerative therapy due to specific disorders or treatments associated to such disorders, including without limitation, muscle and skeletal disorders, neurologic disorders and other metabolic disorders.
  • the reprogrammed UiPSM compositions are used for the treatment of joint or cartilage, muscle or bone damages.
  • the UiPSM and UiSomitoid may also be used advantageously for the production of, but not restricted to, dermal, muscle or skeletal cells from healthy or diseased patients for screening applications in the pharmaceutical industry. Such screening tests can be used to search for new drugs with clinical applications or for toxicology tests.
  • the UiPSM and UiSomitoid may also be used for regenerating bone tissue.
  • the reprogrammed UiPSM and UiSomitoids may also be used for regenerating neuronal tissue, for example in patient suffering from neurodegenerative disorders.
  • methods for treating the condition may include transplanting or grafting a population of UiPSMs or UiSomitoid (such as those described above) onto or in 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 development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
  • treatment of the condition may involve engraftment or transplantation of a population of UiPSM cells and UiSomitoid.
  • An appropriate grafting or transplantation method may be selected depending on where (e.g., what type of tissue or organ) the population of cells are to be transplanted or grafted.
  • an implantable or injectable graft may be used to treat the condition.
  • An implantable graft may include a solid matrix that allows UiPSM cells to be seeded with necessary growth factors (e.g., the environment specific to the population of cells to be implanted) , cultured, and then implanted into the subject in the tissue or organ subject to the condition.
  • An injectable graft which can fill any deficit shape or space in a damaged organ or tissue.
  • the injectable graft involved injection of the UiPSM cells or reprogrammed somatic cells in a cell suspension containing biomaterials that solidifies in situ by virtue of various crosslinking methods known in the art.
  • the mixture may be injected directly into a tissue or organ or may be exposed or glued to the surface of the tissue or organ.
  • Non-limiting examples of biomaterials that can be used in injectable grafts include, but are not limited to, inorganic, natural materials like chitosan, alginate, hylauronic acid, fibrin, gelatin, as well as many synthetic polymers. Such materials are often solidified through methods including thermal gelation, photo cross-linking, or chemical cross-linking.
  • the cell suspension may also be supplemented with soluble signals or specific matrix components. Since these grafts can be relatively easily injected into a target area, there is no (or minimal) need for invasive surgery, which reduces cost, patient discomfort, risk of infection, and scar formation.
  • Chemically modified HA may also be used for injectable material for tissue engineering due to its long-lasting effect while maintaining biocompatibility. Cross-linking methods also maintain the material biocompatibility, and its presence in extensive areas of regenerative or stem/progenitor niches make it an attractive injectable material.
  • kits for A reprogramming system for inducing urine cells comprising:
  • reagents for reconstituting the medium for incuding the urine cells including Advanced DMEM/F12, CHIR99021, EPZ5676, bFGF and EGF;
  • c) optionally, tools for picking epithelial-like cells, such as long glass pasteur straw.
  • kits for rational drug design comprising reprogrammed cells and cells derived or differentiated therefrom obtained by a process of the invention.
  • the kit comprises UiSomitoids and instructions for their use in drug screening in disease models.
  • the kit comprises UiPSM cells, culture medium for cell expansion, and instructions for their use.
  • Example 1 Reprogramming urine cells to urine-derived presomitic mesoderm progenitor cells (UiPSM)
  • pEP4-EO2S-ET2K plasmid was purchased from Addgene (Cat No.: 20927) .
  • the plasmid comprises a pCEP4 backbone and expresses Oct4, Sox2, SV40LT and Klf4.
  • pCEP4-miR-302-367 plasmid by incorporating MicroRNA cluster 302-367 into the pCEP4 backbone (prepared as described in Liao et al., MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem 286, 17359-17364, 2011; Wang et al., Generation of integration-free neural progenitor cells from cells in human urine. Nat Methods 10, 84-89, 2013) .
  • UC Living urine cells
  • REGM Longza, CC-3190
  • epithelial-like cells (above 1.5x10 6 cells) were next pretreated via electroporation with vectors pEP4EO2SET2K (6 ⁇ g) and pCEP4-miR-302-367 (4 ⁇ g) to make the cells susceptible to induction, and then cultured for two days on the REGM medium to recover their ability (Day0 in Fig. 1b) .
  • the pretreated epithelial-like cells were induced for 9 days in a basal medium (Advanced DMEM/F12) supplemented with WNT agonist CHIR99021 (3 ⁇ M; synthesized in-house) , DOT1L inhibitor EPZ5676 (5 ⁇ M; Selleck Chemicals, S7062) , bFGF (5ng/ ⁇ l; PeproTech, P09038) and EGF (5ng/ ⁇ l; R&D systems, 236-EG) .
  • PSM presomitic mesoderm
  • DM defined medium
  • the culturing medium is also referred to as UiPSM maintain medium or “DM medium” .
  • UiPSM cells were seeded at 1 ⁇ 10 5 cells/well (24 well plate) and passaged every 5 days.
  • UiPSM cells could expand rapidly in the UiPSM maintain medium. Similar to the result presented above, Q-PCR, Immunofluorescence and bulk RNA-seq supported that passaged UiPSM stably expressed presomitic markers.
  • UiPSMs derived from the urine cells of Donor 1, Donor 2 and Donor 3 were named as UiPSM1, UiPSM2 and UiPSM3, respectively. Immunofluorescence analysis showed that the continuously passaged UiPSM cells (e.g.
  • Example 2 Differences between urine cell derived PSC (pluripotent stem cell, UiPSC) and PSM (presomitic mesodermal progenitor cell, UiPSM)
  • UiPSC and UiPSM were compared via teratoma experiment by forming teratomas in mice and examining them for representative tissues of all three germ layers.
  • UiPSC cells reprogrammed from urine cells were obtained by strictly following the published protocol (Zhou et al., Generation of human induced pluripotent stem cells from urine samples. Nat Protoc 7, 2080-2089, 2012) .
  • UiPSM derived tissue block was significantly smaller than UiPSC derived tissue block ( Figure 3a) .
  • Paraffin embedding and HE staining were performed next (Figure 3b, panels a-c represent UiPSC, and panels d-f represent UiPSM) .
  • the result showed that UiPSM colonies reduced the risk of tumor formation.
  • RNA seq we profiled single-cell RNA seq to analyze cell composition of tissue derived from UiPSC and UiPSM respectively.
  • GO term analysis show that UiPSM are significantly consistent with muscle development, and GO term further analyze subclusters and show that these subclusters are mainly involved in mesodermal lineage fate like muscle, cartilage, bone, cardiac and limb morphogenesis (Figure 3c-3g) , which supported that UiPSM possess the potential to give rise to cells of the mesoderm lineage, distinct from those of UiPSCs.
  • UiPSMs were digested into single cells using 0.25%Trypsin-EDTA (Gibco, 25200056) and sparsely passaged 7.5x10 4 cells onto new Matrigel-coated cell 24-well plate in UiPSM maintain medium overnight.
  • UiPSM was initially induced via a modified approach including two stages. At stage I, differentiation with medium defined SM was conducted for 15 days, media was changed every 2 days for this step; at stage II, differentiation with medium defined DiKHI was conducted until skeletal muscle fiber bundles are fully present, media was changed every 3 days for this step ( Figure 4a) .
  • SM DMEM/F12 supplemented with 15%KSR (Gibco, 10828028) , 1%ITS (Gibco, 41400045) , 1%NEAA, 0.1 ⁇ M ⁇ -ME, 4 ng/ml IGF-1 (Pepro Tech, 250-19) , 10ng/ml HGF (R&D systems, 294-HG-250) , 3 ⁇ M CHIR99021, 50ng/ml VC (Sigma-Aldrich, 49752) , 0.5ng/ml Dex (Target Mol, T0947L) and 2nM SB431542.
  • DiKHI DMEM supplemented with 15%KSR, 2%Horse serum (Gibco, 16050122) , 1%NEAA, 0.1 ⁇ M ⁇ -ME, 4 ng/ml IGF-1, and 10 ng/ml HGF.
  • UiPSMs were passaged 7.5x10 4 cells onto new Matrigel-coated cell 24-well plate overnight.
  • DMEM basal medium supplemented with 10%FBS (NTC, SFBE) , 50ng/ml VC, 100 nM ⁇ -Glycerophosphate (PeproTech, 154804-51-0) and 1 ⁇ M 1-Thioglycerol (Sigma, 96-27-5) was used for differentiating UiPSM for 15 days, media was changed every 3 days.
  • UiPSMs were passaged 7.5x10 4 cells onto new Matrigel-coated cell 24-well plate overnight.
  • DMEM basal medium supplemented with 10%FBS, 1%ITS, 1%Sodium Pyruvate (Gibco, 11360) , 50ng/ml VC, 0.1 nM ⁇ -Glycerophosphate, 4 ng/mL TGF- ⁇ 3 (PeproTech, 100-36E) and 20ng/ml BMP2 (PeproTech, 500-P195) was used for differentiating UiPSM for 15 days, media was changed every 3 days.
  • UiPSM can differentiate into chrondroblast and osteoblast within 15 days ( Figure 4e, 4h) .
  • Q-PCR confirmed the result by using markers for chrondroblast like ACNA, COL2A1, SOX9, COL9A1 ( Figure 4f) and osteoblast like BMP2, RUNX2, BGLAP, SP7 (Fiugre 4i) .
  • the chrondroblast and osteoblast cells further can be identified with staining by alcian blue (GENMED, GMS80015.1) and alizarin red (GENMED, GMS80017.1) .
  • RNA-seq data analysis confirmed the identity of osteoblast and chrondroblast cells based on associated genes expression (Figure 4g, 4j) . Together, these results suggest that UiPSM, like presomitic mesoderm in vivo, can differentiate into skeletal muscle cells, osteoblast and chrondroblast cells in vitro.
  • the conditions for somitoid generation are further optimized by the following parameters: cell densities, inhibitors for BMP, WNT, NODAL signaling during aggregation and elongation steps.
  • UiSomitoids display polarized patterns of expression for SOX2/CDX2, SOX2/T, and CDX2/T in pairwise co-staining with antibodies at day 9, suggesting an anterior-posterior organization (Figure 5b) .
  • HES7 and MESP2 researchers engineered pepper aptamers which bind and activate fluorescent dyes HBC (could image diverse RNA species in live cells into UiPSM at two loci, HES7 and MESP2) , allowing us to monitor the oscillations of both RNAs.
  • HES7 and MESP2 expressed periodically in the presomitic mesoderm in a dynamic manner. Indeed, UiSomitoid recapitulates this feature well as HES7-GFP regresses posteriorly along the extending axis ( Figure 5c-5e) .
  • MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem 286, 17359-17364.

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Abstract

La présente invention concerne un procédé de reprogrammation ou d'induction de cellules progénitrices du mésoderme présomitique issues de cellules urinaires, une structure somitoïde constituée par les cellules progénitrices du mésoderme présomitique et ses utilisations.
PCT/CN2022/071579 2022-01-12 2022-01-12 Cellules progénitrices du mésoderme présomitique induit, issues de l'urine humaine, et leurs utilisations WO2023133726A1 (fr)

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PCT/CN2022/071579 WO2023133726A1 (fr) 2022-01-12 2022-01-12 Cellules progénitrices du mésoderme présomitique induit, issues de l'urine humaine, et leurs utilisations

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