Methods of reprogramming cells

Abstract

The present invention relates to the field of biomedicine. More specifically, the present invention relates to a method of reprogramming cells by treating a population of cells with a cocktail of small molecules, inducing it to a multi-lineage primed state, from which a population of cells can be directly induced to a specific cell type.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. More specifically, this invention relates to a method for reprogramming cells, which involves treating a cell population with a mixture of small molecules to induce it into a multi-lineage-initiated state, a cell population in which the cell population can be directly induced into a specific cell type. Background of the Invention

[0002] Somatic cells can be reprogrammed into pluripotency through nuclear transfer to oocytes, delivery of transcription factors, or treatment with a cocktail of chemicals (Gurdon, 1962; Takahashi and Yamanaka, 2006; Hou et al., 2013). These somatic cell reprogramming techniques hold great promise in regenerative medicine due to their provision of an unlimited source of cells. Compared to the other two strategies, chemical reprogramming is easier and may be safer for future applications because it is non-integrative, non-genetically modified, easily standardized and time-regulated, and has lower tumorigenicity (Hou et al., 2013).

[0003] In recent years, the molecular mechanisms of cell dynamics and chemical reprogramming have been investigated. For example, the inventors have discovered a state similar to extraembryonic endoderm (XEN) that links chemical reprogramming to CiPSCs of different somatic cell types (Zhao et al., 2015; Ye et al., 2016). Furthermore, dynamic early embryo-like programming is crucial for the transition from XEN-like states to pluripotent states (Zhao et al., 2018), and the efficiency of CiPSC-induced chemical reprogramming can be greatly enhanced by additional chemical enhancers (e.g., bromodeoxyuridine, retinoic acid agonists, Dolt1L inhibitors, and glycogen synthase kinase 3 inhibitors), or even by induction with chemically defined culture media (Long et al., 2015; Zhao et al., 2015; Li et al., 2017b; Cao et al., 2018).

[0004] However, current chemical reprogramming strategies are inefficient and time-consuming, hindering their practical application in the biomedical field. Therefore, there is still a need in this field for more accurate and convenient chemical reprogramming methods. Invention Summary

[0005] In one aspect, the present invention relates to a method for reprogramming cells, the method comprising exposing a cell population to a mixture of small molecules that induce reprogramming for a period of time, thereby obtaining an induced cell population.

[0006] The reprogramming-inducing small molecule mixture mentioned above includes at least a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator.

[0007] The time period mentioned therein is no more than approximately 11 days.

[0008] In one aspect, the present invention relates to a method for reprogramming starting cells into target cells, the method comprising a first phase and a second phase.

[0009] The first stage involves exposing a starting cell population to a mixture of small molecules that induce reprogramming for a period of time to obtain an induced cell population. The mixture of small molecules induces reprogramming includes at least a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator. The period of time is no more than 11 days.

[0010] The second stage involves exposing the induced cell population obtained in the first stage to a target cell reprogramming small molecule mixture, thereby obtaining the target cell population. Brief description of the attached diagram

[0011] Figure 1 Chemical mixtures activate endogenous expression of transcription factors across multiple lineages. A. Schematic diagram of RNA-seq analysis strategies during chemical reprogramming. B. Variable patterns of gene expression in the early stages of chemical reprogramming. C. Representative genes from clusters 1 / 2, 3 / 4 / 5, 6 / 7 / 8 / 9, and 10 in a large RNA spectrum. D. Activated endogenous transcription factors associated with different embryonic layers during the first 8 days of chemical reprogramming. E. Number of activated transcription factors in three different XEN reprogramming mixtures. F. GO analysis of upregulated activated genes during the first 8 days (gene grouping in clusters 3, 4, and 5).

[0012] Figure 2 Chemical mixtures activate endogenous expression of transcription factors across multiple lineages. A. Representative GO analysis of clusters 1 / 2, 6 / 7 / 8 / 9, and 10 in a large RNA spectrum. B. Variable patterns of early gene expression mediated by VC6FAE-mediated chemical reprogramming. C. Variable patterns of early gene expression mediated by C6F5UE-mediated chemical reprogramming. D. Cellular priming in multiple cell types. E. GD analysis of endogenous transcription factors activated in NDF and TTF.

[0013] Figure 3Following CaMP, several other cell types have emerged. A. Representative genes of induced neurons detected by immunofluorescence. Scale bar, 100 μm. B. Relative expression of neuronal genes under different conditions detected by RT-qPCR. C. Improvement of neuronal induction with CaMP. D. Representative genes of induced skeletal muscle cells detected by immunofluorescence. Scale bar, 100 μm. E. Relative expression of skeletal muscle genes under different conditions detected by RT-qPCR. F. Improvement of skeletal muscle cell induction with CaMP. G. RNA-seq profiles of neuron-specific genes induced by C6FI with or without the CaMP step. Quantile normalization was performed on different arrays, and then the expression profiles of each gene were normalized using Z-Score. H. RNA-seq profiles of skeletal muscle-specific genes induced by C6FS with or without the CaMP step. I. PCA projection of skeletal muscle and neuronal reprogramming processes. J. Schematic diagram of the induction of functional cells from induced cells.

[0014] Figure 4 Following CaMP, several other cell types emerged. A. Number of neurons induced by different pretreatments. B. Effect of removing chemicals from C6FI on neuronal induction. C. Effect of Sall4 and Gata4 knockdown on neurons. D. Number of skeletal muscle cells induced by different pretreatments. E. Effect of removing chemicals from C6FS on skeletal muscle cell induction. F. Effect of Sall4 and Gata4 knockdown on skeletal muscle induction. G. RT-qPCR of Gata4 expression after Gata4 knockdown. H. RT-qPCR of Sall4 expression after Sall4 knockdown.

[0015] Figure 5 Random activation of endogenous transcription factors by chemical mixtures. A. t-SNE projection of all 610 monomeric cells during reprogramming. B. Examples of lineage-specific genes activated during the t-SNE projection. C. Statistics on the number of activated and inactivated genes in the first 5 days. D. Correlation among activated transcription factors during days 1-5. E. Statistics on correlation coefficients of transcription factor pairs. F. Expression of major XEN genes (Sox17, Sal4, Gata4) and major MEF genes (Osr1, Prrx1, Twist2) during the t-SNE projection.

[0016] Figure 6Random activation of endogenous transcription factors by chemical mixtures. A. Schematic diagram of early chemical reprogramming single-cell RNA-seq time points. B. Quality control of raw single-cell sequencing data. C. Number of genes detected at each time point. D. Number of cells detected at each time point. E. PCA projection of all 610 single cells during reprogramming. F. Number of activated transcription factors in each cell during the first 6 days. Colors indicate different time points. G. Expression levels of early activated transcription factors on day 20.

[0017] Figure 7 Sox17 activates progressive cell fate specialization and switching in cells. A. Analysis of MEF and XEN properties in t-SNE projections. B. Differentially expressed genes between trapped and successful branches. C. Subset relationships between Sox17 and other major XEN genes revealed by immunostaining. Scale bar, 100 micrometers. D. Relative expression of XEN genes after Sox17 knockdown. E. Mathematical modeling of stable and unstable states of the XEN gene regulatory network. F. Transcription factor-related networks of XEN and MEF. G. Relative expression of MEF genes with Sox17 overexpression. H. Relative expression of XEN genes after MEF knockdown. OPT = Osr1, Prrx1, and Twist2.

[0018] Figure 8 Sox17 activates progressive cell fate specialization and transition in cells. A. t-SNE map of a single cell with clustering information. B. Gene expression profile of each cluster in the t-SNE map. C. Dynamic changes in MEF and XEN properties. D. Schematic diagram of cell pathways through trapped and successful branches. E. Number of XEN communities generated after cell passage through trapped and successful branches. F. Expression levels of major XEN genes increasing with XEN properties. G. Relative expression of XEN genes with Sox17 overexpression. H. Mimic gene expression with different initial Sox17 expression. I. Relative expression of genes in fibroblasts with MEF knockdown. OPT = Osr1, Prrx1, and Twist2.

[0019] Figure 9 Chemical enhancers are beneficial at different stages of reprogramming. A. Effects of CH55 and VPA on XEN genes at different time points. Compare C6FAE conditions with additional CH55 or VPA to C6FAE conditions only. Red indicates improvement. B. Effects of CH55 and VPA on the similarity of genes and lineages at different time points. C. Relative expression of XEN genes after CH55 treatment for different time points. D. Relative expression of XEN genes after VPA treatment for different time points. E. Relative number of XEN communities after CH55 / VPA treatment for different time points.

[0020] Figure 10 Chemical enhancers are beneficial for different stages of reprogramming. A. Morphology and gene expression in fuzzy and smoothed communities. Scale bar, 100 micrometers. B. Total community number and XEN-like community number in different chemical combinations. C. Relative expression of the major XEN gene in fuzzy and smoothed communities. D. Relative expression of the major MEF gene in fuzzy and smoothed communities. E. Relative expression of XEN gene after treatment with CH55 for different time periods. F. Relative expression of XEN gene after treatment with VPA for different time periods. G. Relative changes in XEN gene after treatment with CH55 / VPA for different time periods.

[0021] Figure 11 The core reprogramming chemical (FC6) simultaneously initiates cell plastination and XEN cell specialization through different mechanisms. A. Number of transcription factors activated by CHIR99021 / Forskolin / 616452 relative to MEF during the first 4 days of XEN reprogramming. The gene list is limited to 150 transcription factors activated by C6FAE. B. Number of transcription factors activated by CHIR99021 / Forskolin / 616452 relative to MEF during the first 8 days of XEN reprogramming. The gene list is limited to 150 transcription factors activated by C6FAE. C. Transcription factors regulated by CHIR99021 / Forskolin / 616452 relative to MEF during the first 4 days of XEN reprogramming. D. Transcription factors regulated by CHIR99021 / Forskolin / 616452 relative to MEF during the first 8 days of XEN reprogramming. E. Relative expression of the XEN major gene on day 12 after removal of CHIR99021 / Forskolin / 616452 from day 4. F. Immunostaining of Sal4 and Gata4 on day 12 after removal of 616452 from day 4. Scale bar, 100 μm. G. Quantification of Gata4-positive colonies on day 12 after removal of CHIR99021 / Forskolin / 616452 from day 4. H. Immunostaining of Sal4 and Gata4 on day 12 after removal of CHIR99021 and Forskolin from day 6. Scale bar, 100 μm. I. Quantification of Sal4-positive colonies on day 12 after removal of CHIR99021 / Forskolin / 616452 from day 6. J. Schematic diagram of the role of CHIR99021 / Forskolin / 616452 in regulating the XEN major gene.

[0022] Figure 12The core reprogramming chemical (FC6) simultaneously initiates cell plasticity and XEN cell specialization through different mechanisms. A. Transcription factors regulated by CHIR99021 / Forskolin / 616452 relative to those regulated by C6FAE in the first 4 days (red dots, upregulated transcription factors; blue dots, downregulated transcription factors; arrows, promotion; horizontal lines, inhibition. Deeper lines: stronger regulation). Upregulated and downregulated genes were detected by comparing the intact mixture with the mixture after removing individual molecules. B. Relative expression of the XEN major gene on day 12 after removing CHIR99021 / Forskolin / 616452 from day 0. C. Relative expression of the XEN gene with Sall4 knockdown. D. Relative expression of the XEN gene with Sall4 expression. E. Relationship between Sall4 and Gata4 at the translational level. Scale bar, 100 micrometers. F. Relative expression of the XEN major gene on day 12 after removing CHIR99021 / Forskolin / 616452 from day 8.

[0023] Figure 13 Multi-lineage induced stage model diagram Invention Details

[0024] I. Methods for reprogramming cells

[0025] The inventors have surprisingly discovered that by briefly treating differentiated cell populations, such as fibroblasts, with a specific mixture of reprogramming small molecules, the cell population can be induced into a state known as multi-lineage induction. This cell population, through further target cell type-specific directed induction, can be directly reprogrammed into the desired target cell type. This method of cell reprogramming eliminates the need for conventional, longer-duration (e.g., an additional 8-20 days) pluripotent stem cell induction or so-called XEN-like cell induction, thus significantly saving time and cost in cell reprogramming. Furthermore, the phased chemically induced reprogramming method of this invention significantly improves the efficiency of reprogramming starting cells into target cells compared to conventional chemical reprogramming methods (e.g., using only a single reprogramming small molecule mixture throughout).

[0026] The present invention relates to a method for reprogramming cells, the method comprising exposing a cell population to a mixture of small molecules that induce reprogramming for a period of time, thereby obtaining an induced cell population, wherein the period of time is no more than about 11 days.

[0027] In some embodiments, the time period is no more than about 10 days, no more than about 9 days, no more than about 8 days, no more than about 7 days, no more than about 6 days, no more than about 5 days, no more than about 4 days, no more than about 3 days, no more than about 2 days, or no more than about 1 day. In some embodiments, the time period is about 1-11 days, for example, about 2-10 days, about 3-9 days, about 4-8 days, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or about 11 days. In some preferred embodiments, the time period is about 4 days.

[0028] In some embodiments, the reprogramming-initiating small molecule mixture includes at least a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator.

[0029] Examples of GSK-3α / β inhibitors include, but are not limited to, CHIR99021 or its structural analogues, BIO or its structural analogues, IM-12 or its structural analogues. In some preferred embodiments, the GSK-3α / β inhibitor is CHIR99021 or its structural analogue.

[0030] Examples of cAMP activators include, but are not limited to, Forskolin or its structural analogs, IBMX or its structural analogs, Dibutyryl-cAMP or its structural analogs. In some preferred embodiments, the cAMP activator is Forskolin or its structural analogs.

[0031] Examples of TGFβR-1 / ALK5 inhibitors include, but are not limited to, 616452 or its structural analogues, SB431542 or its structural analogues, and LY364947 or its structural analogues. In some preferred embodiments, the TGFβR-1 / ALK5 inhibitor is 616452 or its structural analogues, etc.

[0032] In some embodiments, the method does not include the step of exposing the cell population to exogenous transcription factors. For example, the method does not include exposing the cell population to exogenous transcription factors related to cell reprogramming, such as Oct4, Sox2, Klf4, and C-Myc. That is, the method for reprogramming cells of the present invention is a chemically induced (using small molecule compounds) reprogramming method.

[0033] Unless explicitly limited to the opposite, "cell" includes any somatic cell, embryonic stem (ES) cell, adult stem cell, organ-specific stem cell, nuclear transfer (NT) unit, and stem-like cell. Cells can be obtained from any organ or tissue. Cells can be human or from other animals. For example, cells can be mouse, guinea pig, rat, cow, horse, pig, sheep, goat, etc. Cells can also originate from non-human primates.

[0034] In one embodiment, the cells in the cell population are differentiated cells. In one embodiment, the cells in the cell population are mesodermal-derived cells such as cardiac cells, ectodermal-derived cells such as nerve cells, and endoderm-derived cells such as colon cells. In one embodiment, the cells are neurons, skeletal muscle cells, hepatocytes, fibroblasts, osteoblasts, chondrocytes, adipocytes, endothelial cells, interstitial cells, smooth muscle cells, cardiomyocytes, nerve cells, hematopoietic cells, pancreatic islet cells, or virtually any cell in the body. In one embodiment, the cells are fibroblasts.

[0035] "Differentiation" refers to the process by which cells specialize in structure and function during embryonic development. During the development of multicellular organisms, different cells and tissues acquire different gene expression patterns; that is, each cell type has a unique gene expression pattern, which is generally considered to become "fixed" and unchanging after cell differentiation. "Differentiated cells" refer to cell groups that gradually develop from cells of the same origin, exhibiting different morphological structures and functional characteristics.

[0036] In this article, the terms "initiated cell population" and "multi-lineage induced cell population" are used interchangeably, referring to a cell population containing multiple cell types, each with the potential to develop into a specific cell type. Such induced cell populations exhibit gene transcription patterns different from the initial differentiated cell population; for example, many lineage-related transcription factors (TFs) are upregulated in induced cell populations, and endogenous developmentally relevant transcription factors are expressed randomly. "Multi-lineage induced" by small molecule compounds is also known as "chemically activated multi-lineage induced (CaMP)."

[0037] In one embodiment, the reprogramming-initiating small molecule mixture further comprises a RARα activator and / or a DOT1L inhibitor.

[0038] Examples of the RARα activator include, but are not limited to, AM580 or its structural analogues, Tamibarotene or its structural analogues, TTNPB or its structural analogues. In some preferred embodiments, the RARα activator is AM580 or its structural analogue.

[0039] Examples of DOT1L inhibitors include, but are not limited to, EPZ00477 or its structural analogues, SGC0496 or its structural analogues, EPZ5676 or its structural analogues, etc. In some preferred embodiments, the DOT1L inhibitor is EPZ00477 or its structural analogues.

[0040] In one embodiment, the reprogramming-initiating small molecule mixture further comprises an HDAC inhibitor, a histone lysine methyltransferase (HMTase) inhibitor (e.g., UNC0638 or its structural analogues, BIX01294 or its structural analogues, UNC0642 or its structural analogues), and / or a RAR-α / β activator (e.g., CH55 or its structural analogues).

[0041] The HDAC inhibitors include, but are not limited to, valproic acid (VPA) or its structural analogs, Trichostatin A (TSA) or its structural analogs, Voronostat (SAHA) or its structural analogs. In some preferred embodiments, the HDAC inhibitor is valproic acid (VPA) or its structural analogs.

[0042] In one embodiment, the reprogramming-initiating small molecule mixture comprises a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator. In another embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021 or a structural analog thereof, Forskolin or a structural analog thereof, and 616452 or a structural analog thereof. In a specific embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021, Forskolin, and 616452.

[0043] In one embodiment, the reprogramming-initiating small molecule mixture comprises a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, a cAMP activator, a RARα activator, and a DOT1L inhibitor. In another embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021 or a structural analog thereof, Forskolin or a structural analog thereof, 616452 or a structural analog thereof, AM580 or a structural analog thereof, and EPZ00477 or a structural analog thereof. In a specific embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021, Forskolin, 616452, AM580, and EPZ00477.

[0044] In one specific embodiment, the small molecule mixture that initiates reprogramming comprises VPA, CHIR99021, Forskolin, 616452, AM580, and EPZ00477. In another specific embodiment, the small molecule mixture that initiates reprogramming comprises CHIR99021, Forskolin, 616452, EPZ00477, UNC0638, and CH55. In yet another specific embodiment, the small molecule mixture that initiates reprogramming comprises CHIR99021, Forskolin, 616452, and CH55.

[0045] In one embodiment, the concentration of CHIR99021 used is from about 1 μM to about 100 μM, for example, about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, and about 100 μM. Preferably, the concentration of CHIR99021 used is about 20 μM.

[0046] In one embodiment, the concentration of 616452 used is between approximately 1 μM and 50 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, and approximately 50 μM. Preferably, the concentration of 616452 used is approximately 10 μM.

[0047] In one embodiment, the concentration of Forskolin used is from about 10 μM to about 200 μM, for example, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM, and about 200 μM. Preferably, the concentration of Forskolin used is about 50 μM.

[0048] In one embodiment, the concentration of AM580 used is approximately 0.01 μM to approximately 1 μM, for example, approximately 0.01 μM, approximately 0.02 μM, approximately 0.03 μM, approximately 0.04 μM, approximately 0.05 μM, approximately 0.06 μM, approximately 0.07 μM, approximately 0.08 μM, approximately 0.09 μM, approximately 0.1 μM, approximately 0.2 μM, approximately 0.3 μM, approximately 0.4 μM, approximately 0.5 μM, approximately 0.6 μM, approximately 0.7 μM, approximately 0.8 μM, approximately 0.9 μM, or approximately 1 μM. Preferably, the concentration of AM580 used is approximately 0.05 μM.

[0049] In one embodiment, the concentration of EPZ004777 used is approximately 1 μM to approximately 20 μM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 12.5 μM, approximately 15 μM, approximately 17.5 μM, and approximately 20 μM. Preferably, the concentration of EPZ004777 used is approximately 5 μM.

[0050] In one embodiment, the concentration of VPA is about 0.1 mM to about 0.9 mM, for example about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, or about 0.9 mM. Preferably, the concentration of VPA is about 0.5 mM.

[0051] In one embodiment, the concentration of CH55 is about 0.2 μM, about 0.5 μM, about 1 μM, about 2 μM, about 5 μM, or between about 0.5 and about 2 μM. Preferably, the concentration of CH55 is about 1 μM.

[0052] In one embodiment, the concentration of UNC0638 is approximately 0.1 μM, approximately 0.2 μM, approximately 0.5 μM, approximately 1 μM, approximately 2 μM, or between approximately 0.2 and approximately 2 μM. Preferably, the concentration of UNC0638 is approximately 0.5 μM.

[0053] In another aspect, the present invention also covers induced cells obtained by the method of reprogramming cells of the present invention and their uses.

[0054] II. Methods for reprogramming starting cells into target cells

[0055] This invention relates to a method for reprogramming starting cells into target cells, the method comprising a first stage and a second stage.

[0056] The first stage involves exposing a starting cell population to a mixture of small molecules that induce reprogramming for a period of time, wherein the period of time is no more than 11 days.

[0057] The second stage involves exposing the induced cell population obtained in the first stage to a target cell reprogramming small molecule mixture, thereby obtaining the target cell population.

[0058] In some embodiments, the duration of the first phase is no more than approximately 10 days, no more than approximately 9 days, no more than approximately 8 days, no more than approximately 7 days, no more than approximately 6 days, no more than approximately 5 days, no more than approximately 4 days, no more than approximately 3 days, no more than approximately 2 days, or no more than approximately 1 day. In some embodiments, the duration of the first phase is approximately 1-11 days, for example, approximately 2-10 days, approximately 3-9 days, approximately 4-8 days, for example, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or approximately 11 days. In some preferred embodiments, the duration of the first phase is approximately 4 days.

[0059] In some embodiments, the reprogramming-initiating small molecule mixture includes at least a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator.

[0060] Examples of GSK-3α / β inhibitors include, but are not limited to, CHIR99021 or its structural analogues, BIO or its structural analogues, IM-12 or its structural analogues. In some preferred embodiments, the GSK-3α / β inhibitor is CHIR99021 or its structural analogue.

[0061] Examples of cAMP activators include, but are not limited to, Forskolin or its structural analogs, IBMX or its structural analogs, Dibutyryl-cAMP or its structural analogs. In some preferred embodiments, the cAMP activator is Forskolin or its structural analogs.

[0062] Examples of TGFβR-1 / ALK5 inhibitors include, but are not limited to, 616452 or its structural analogues, SB431542 or its structural analogues, and LY364947 or its structural analogues. In some preferred embodiments, the TGFβR-1 / ALK5 inhibitor is 616452 or its structural analogues, etc.

[0063] In some embodiments, the method does not include the step of exposing the cell population to exogenous transcription factors. For example, the method does not include exposing the cell population to exogenous transcription factors related to cell reprogramming, such as Oct4, Sox2, Klf4, and C-Myc. That is, the method for reprogramming cells of the present invention is a chemically induced (using small molecule compounds) reprogramming method.

[0064] In one embodiment, the reprogramming-initiating small molecule mixture further comprises a RARα activator and / or a DOT1L inhibitor.

[0065] Examples of the RARα activator include, but are not limited to, AM580 or its structural analogues, Tamibarotene or its structural analogues, TTNPB or its structural analogues. In some preferred embodiments, the RARα activator is AM580 or its structural analogue.

[0066] Examples of DOT1L inhibitors include, but are not limited to, EPZ00477 or its structural analogues, SGC0496 or its structural analogues, EPZ5676 or its structural analogues, etc. In some preferred embodiments, the DOT1L inhibitor is EPZ00477 or its structural analogues.

[0067] In one embodiment, the reprogramming-initiating small molecule mixture further comprises an HDAC inhibitor, UNC0638 or a structural or functional analogue thereof, and / or CH55 or a structural or functional analogue thereof.

[0068] The HDAC inhibitors include, but are not limited to, valproic acid (VPA) or its structural analogs, Trichostatin A (TSA) or its structural analogs, Voronostat (SAHA) or its structural analogs. In some preferred embodiments, the HDAC inhibitor is valproic acid (VPA) or its structural analogs.

[0069] In one embodiment, the reprogramming-initiating small molecule mixture comprises a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator. In another embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021 or a structural analog thereof, Forskolin or a structural analog thereof, and 616452 or a structural analog thereof. In a specific embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021, Forskolin, and 616452.

[0070] In one embodiment, the reprogramming-initiating small molecule mixture comprises a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, a cAMP activator, a RARα activator, and a DOT1L inhibitor. In another embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021 or a structural analog thereof, Forskolin or a structural analog thereof, 616452 or a structural analog thereof, AM580 or a structural analog thereof, and EPZ00477 or a structural analog thereof. In a specific embodiment, the reprogramming-initiating small molecule mixture comprises CHIR99021, Forskolin, 616452, AM580, and EPZ00477.

[0071] In one specific embodiment, the small molecule mixture that initiates reprogramming comprises VPA, CHIR99021, Forskolin, 616452, AM580, and EPZ00477. In another specific embodiment, the small molecule mixture that initiates reprogramming comprises CHIR99021, Forskolin, 616452, EPZ00477, UNC0638, and CH55. In yet another specific embodiment, the small molecule mixture that initiates reprogramming comprises CHIR99021, Forskolin, 616452, and CH55.

[0072] In one embodiment, the concentration of CHIR99021 used is from about 1 μM to about 100 μM, for example, about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, and about 100 μM. Preferably, the concentration of CHIR99021 used is about 20 μM.

[0073] In one embodiment, the concentration of 616452 used is between approximately 1 μM and 50 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, and approximately 50 μM. Preferably, the concentration of 616452 used is approximately 10 μM.

[0074] In one embodiment, the concentration of Forskolin used is from about 10 μM to about 200 μM, for example, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM, and about 200 μM. Preferably, the concentration of Forskolin used is about 50 μM.

[0075] In one embodiment, the concentration of AM580 used is approximately 0.01 μM to approximately 1 μM, for example, approximately 0.01 μM, approximately 0.02 μM, approximately 0.03 μM, approximately 0.04 μM, approximately 0.05 μM, approximately 0.06 μM, approximately 0.07 μM, approximately 0.08 μM, approximately 0.09 μM, approximately 0.1 μM, approximately 0.2 μM, approximately 0.3 μM, approximately 0.4 μM, approximately 0.5 μM, approximately 0.6 μM, approximately 0.7 μM, approximately 0.8 μM, approximately 0.9 μM, or approximately 1 μM. Preferably, the concentration of AM580 used is approximately 0.05 μM.

[0076] In one embodiment, the concentration of EPZ004777 used is approximately 1 μM to approximately 20 μM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 12.5 μM, approximately 15 μM, approximately 17.5 μM, and approximately 20 μM. Preferably, the concentration of EPZ004777 used is approximately 5 μM.

[0077] In one embodiment, the concentration of VPA used is from about 0.1 mM to about 0.9 mM, for example, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, or about 0.9 mM. Preferably, the concentration of VPA used is about 0.5 mM.

[0078] In one embodiment, the concentration of CH55 is about 0.2 μM, about 0.5 μM, about 1 μM, about 2 μM, about 5 μM, or between about 0.5 and about 2 μM. Preferably, the concentration of CH55 is about 1 μM.

[0079] In one embodiment, the concentration of UNC0638 is approximately 0.1 μM, approximately 0.2 μM, approximately 0.5 μM, approximately 1 μM, approximately 2 μM, or between approximately 0.2 and approximately 2 μM. Preferably, the concentration of UNC0638 is approximately 0.5 μM.

[0080] In one embodiment, the starting cell is a differentiated cell. In one embodiment, the starting cell can be a mesodermal-derived cell such as a heart cell, an ectodermal-derived cell such as a nerve cell, or an endoderm-derived cell such as a colon cell. In one embodiment, the starting cell can be a neuron, skeletal muscle cell, hepatocyte, fibroblast, osteoblast, chondrocyte, adipocyte, endothelial cell, mesenchymal cell, smooth muscle cell, cardiomyocyte, nerve cell, hematopoietic cell, pancreatic islet cell, or virtually any cell in the body. In one embodiment, the starting cell is a fibroblast.

[0081] In one embodiment, the target cell is of a different cell type than the initiating cell. In one embodiment, the target cell is a mesodermal-derived cell such as a heart cell, an ectodermal-derived cell such as a nerve cell, or an endoderm-derived cell such as a colon cell. In one embodiment, the target cell is a neuron, skeletal muscle cell, hepatocyte, fibroblast, osteoblast, chondrocyte, adipocyte, endothelial cell, interstitial cell, smooth muscle cell, cardiomyocyte, nerve cell, hematopoietic cell, pancreatic islet cell, or virtually any cell in the body.

[0082] As used herein, "target cell reprogramming small molecule mixtures" refer to small molecule mixtures capable of directionally inducing pluripotent cells into specific cell types. Various small molecule mixtures are known in the art that can directionally induce, for example, CiPSCs and XEN-like cells into specific cell types such as neurons, cardiomyocytes, and skeletal muscle cells. These small molecule mixtures can be used, or modified for use in this invention.

[0083] In some embodiments, the types of small molecules in the "target cell reprogramming small molecule mixture" may be the same as or different from those in the "initiating reprogramming small molecule mixture". In some embodiments, the amount of one or more small molecules in the "target cell reprogramming small molecule mixture" differs from the amount of the corresponding small molecules in the "initiating reprogramming small molecule mixture".

[0084] In some embodiments, the target cell reprogramming small molecule mixture includes at least a GSK-3α / β inhibitor, a TGFβR-1 / ALK5 inhibitor, and a cAMP activator. In some specific embodiments, the target cell reprogramming small molecule mixture includes at least CHIR99021 or a structural analog thereof, Forskolin or a structural analog thereof, and 616452 or a structural analog thereof. In one embodiment, the target cell reprogramming small molecule mixture further includes an HDAC inhibitor. The HDAC inhibitor includes, but is not limited to, valproic acid (VPA) or a structural analog thereof, Trichostatin A (TSA) or a structural analog thereof, Voronostat (SAHA) or a structural analog thereof. In some preferred embodiments, the HDAC inhibitor is valproic acid (VPA) or a structural analog thereof. In some preferred embodiments, the HDAC inhibitor is valproic acid (VPA) or a structural analog thereof. In one embodiment, the target cell reprogramming small molecule mixture further includes a synthesis promoter of adult neurogenesis, such as ISX9 or a structural analog thereof. In one embodiment, the target cell reprogramming small molecule mixture further comprises SB431542 or a structural analogue thereof.

[0085] In one embodiment, the concentration of CHIR99021 used in the second stage is approximately 1 μM to approximately 100 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, and approximately 100 μM. Preferably, the concentration of CHIR99021 used is approximately 20 μM.

[0086] In one embodiment, the concentration of 616452 used in the second stage is between approximately 1 μM and 50 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, and approximately 50 μM. Preferably, the concentration of 616452 used is approximately 10 μM.

[0087] In one embodiment, the concentration of Forskolin used in the second stage is approximately 10 μM to approximately 200 μM, for example, approximately 10 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, approximately 100 μM, approximately 125 μM, approximately 150 μM, approximately 175 μM, or approximately 200 μM. Preferably, the concentration of Forskolin used is approximately 50 μM.

[0088] In one embodiment, the concentration of VPA used in the second stage is approximately 0.1 mM to approximately 1 mM, for example, approximately 0.1 mM, approximately 0.2 mM, approximately 0.3 mM, approximately 0.4 mM, approximately 0.5 mM, approximately 0.6 mM, approximately 0.7 mM, approximately 0.8 mM, approximately 0.9 mM, or approximately 1 mM. Preferably, the concentration of VPA used is approximately 0.5 mM.

[0089] In one embodiment, the concentration of SB431542 used in the second stage is approximately 1 μM to approximately 5 μM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, or approximately 5 μM. Preferably, the concentration of SB431542 is 3 μM.

[0090] In one embodiment, the concentration of ISX-9 used in the second stage is from about 1 μM to about 20 μM, for example, about 1 μM, about 2.5 μM, about 5 μM, about 7.5 μM, about 10 μM, about 12.5 μM, about 15 μM, about 17.51 ​​μM, or about 20 μM. Preferably, the concentration of ISX-9 is 10 μM.

[0091] Without any theoretical limitations, it is believed that the induced cell population generated in the first stage of the method of the present invention already contains a variety of different cell types, each with the potential or cell fate to develop into different specific cell types. Therefore, in the second stage, through targeted treatment of the target cell reprogramming small molecule mixture, cells with the desired target cell fate can be enriched and expanded, thereby obtaining the desired target cell population.

[0092] In one embodiment, the target cells are neuronal cells, and the target cell reprogramming small molecule mixture comprises CHIR99021, Forskolin, 616452, and ISX9. In one embodiment, CHIR99021 is used at a concentration of 20 μM, 616452 at a concentration of 10 μM, Forskolin at a concentration of 50 μM, and ISX9 at a concentration of 10 μM.

[0093] In one embodiment, the target cells are skeletal muscle cells, and the target cell reprogramming small molecule mixture comprises CHIR99021, Forskolin, 616452, and SB431542. In one embodiment, CHIR99021 is used at a concentration of 20 μM, 616452 at a concentration of 10 μM, Forskolin at a concentration of 50 μM, and SB431542 at a concentration of 3 μM.

[0094] In one embodiment, in the second phase, the induced cells are exposed to the target cell reprogramming small molecule mixture for a period of time, said period of time being no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, or about 8-12 days.

[0095] In another aspect, the present invention also covers target cells obtained by the method of the present invention and their uses.

[0096] III. Composition and Culture Medium

[0097] This invention relates to an initiation reprogramming composition for preparing "multi-lineage initiated cell populations", the composition comprising an initiation reprogramming small molecule mixture as defined above.

[0098] This invention relates to an initiation reprogramming medium for preparing "multi-lineage initiated cell populations", the medium comprising an initiation reprogramming small molecule mixture as defined above.

[0099] In one embodiment, the concentration of CHIR99021 in the initiation reprogramming medium is approximately 1 μM to approximately 100 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, or approximately 100 μM. Preferably, the concentration of CHIR99021 is approximately 20 μM.

[0100] In one embodiment, the concentration of 616452 in the initiation reprogramming medium is between approximately 1 μM and 50 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, or approximately 50 μM. Preferably, the concentration of 616452 is approximately 10 μM.

[0101] In one embodiment, the concentration of Forskolin in the initiation reprogramming medium is approximately 10 μM to approximately 200 μM, for example, approximately 10 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, approximately 100 μM, approximately 125 μM, approximately 150 μM, approximately 175 μM, or approximately 200 μM. Preferably, the concentration of Forskolin is approximately 50 μM.

[0102] In one embodiment, the concentration of AM580 in the initiation reprogramming medium is approximately 0.01 μM to approximately 1 μM, for example, approximately 0.01 μM, approximately 0.02 μM, approximately 0.03 μM, approximately 0.04 μM, approximately 0.05 μM, approximately 0.06 μM, approximately 0.07 μM, approximately 0.08 μM, approximately 0.09 μM, approximately 0.1 μM, approximately 0.2 μM, approximately 0.3 μM, approximately 0.4 μM, approximately 0.5 μM, approximately 0.6 μM, approximately 0.7 μM, approximately 0.8 μM, approximately 0.9 μM, or approximately 1 μM. Preferably, the concentration of AM580 is approximately 0.05 μM.

[0103] In one embodiment, the concentration of EPZ004777 in the initiation reprogramming medium is approximately 1 μM to approximately 20 μM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 12.5 μM, approximately 15 μM, approximately 17.5 μM, or approximately 20 μM. Preferably, the concentration of EPZ004777 is approximately 5 μM.

[0104] In one embodiment, the concentration of VPA in the initiation reprogramming medium is about 0.1 mM to about 0.9 mM, for example, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, or about 0.9 mM. Preferably, the concentration of VPA is about 0.5 mM.

[0105] In one embodiment, the concentration of CH55 is about 0.2 μM, about 0.5 μM, about 1 μM, about 2 μM, about 5 μM, or between about 0.5 and about 2 μM. Preferably, the concentration of CH55 is about 1 μM.

[0106] In one embodiment, the concentration of UNC0638 is approximately 0.1 μM, approximately 0.2 μM, approximately 0.5 μM, approximately 1 μM, approximately 2 μM, or between approximately 0.2 and approximately 2 μM. Preferably, the concentration of UNC0638 is approximately 0.5 μM.

[0107] In one embodiment, the initiation reprogramming medium comprises 0.5 mM valproic acid, 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, 0.05 μM AM580, and 5 μM EPZ004777. Alternatively, the initiation reprogramming medium comprises 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, 0.05 μM AM580, and 5 μM EPZ004777. Alternatively, the initiation reprogramming medium comprises 20 μM CHIR99021, 10 μM 616452, and 50 μM Forskolin.

[0108] In one embodiment, the initiation reprogramming medium is KnockOut DMEM supplemented with KnockOut serum substitute (KSR), FBS, GlutaMAX, NEAA (Non-Essential Amino Acid), 2-mercaptoethanol, basic fibroblast growth factor (bFGF), and the initiation reprogramming small molecule mixture. In some embodiments, the initiation reprogramming medium also contains antibiotics such as penicillin and / or streptomycin.

[0109] In one specific embodiment, the initiation reprogramming medium is KnockOut DMEM supplemented with 10% KnockOut serum substitute (KSR), 10% FBS, 1% GlutaMAX, 1% NEAA, 0.055 mM 2-mercaptoethanol, 1% penicillin-streptomycin, 50 ng / ml basic fibroblast growth factor (bFGF), and the initiation reprogramming small molecule mixture.

[0110] This invention relates to a target cell reprogramming composition for preparing a target cell population from a "multi-lineage-initiated cell population," the composition comprising a mixture of target cell reprogramming small molecules. The "mixture of target cell reprogramming small molecules" is as defined above.

[0111] This invention relates to a target cell reprogramming medium for preparing target cell populations from "multi-lineage-initiated cell populations," comprising a mixture of target cell reprogramming small molecules. The "target cell reprogramming small molecule mixture" is as defined above. The target cell reprogramming medium may also additionally contain components suitable for or necessary for target cell growth. For example, the target cell reprogramming medium of this invention may be derived from media known in the art for the culture of specific target cells.

[0112] In some embodiments, the composition of the "target cell reprogramming small molecule mixture" may be the same as or different from that of the "initiating reprogramming small molecule mixture". In some embodiments, the amount of one or more small molecules in the "target cell reprogramming small molecule mixture" differs from the amount of the corresponding small molecules in the "initiating reprogramming small molecule mixture".

[0113] In one embodiment, the concentration of CHIR99021 in the target cell reprogramming medium is approximately 1 μM to approximately 100 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, or approximately 100 μM. Preferably, the concentration of CHIR99021 is approximately 20 μM.

[0114] In one embodiment, the concentration of 616452 in the target cell reprogramming medium is between approximately 1 μM and 50 μM, for example, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, and approximately 50 μM. Preferably, the concentration of 616452 is approximately 10 μM.

[0115] In one embodiment, the concentration of Forskolin in the target cell reprogramming medium is approximately 10 μM to approximately 200 μM, for example, approximately 10 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, approximately 100 μM, approximately 125 μM, approximately 150 μM, approximately 175 μM, or approximately 200 μM. Preferably, the concentration of Forskolin is approximately 50 μM.

[0116] In one embodiment, the concentration of VPA in the target cell reprogramming medium is approximately 0.1 mM to approximately 1 mM, for example, approximately 0.1 mM, approximately 0.2 mM, approximately 0.3 mM, approximately 0.4 mM, approximately 0.5 mM, approximately 0.6 mM, approximately 0.7 mM, approximately 0.8 mM, approximately 0.9 mM, or approximately 1 mM. Preferably, the concentration of VPA is approximately 0.5 mM.

[0117] In one embodiment, the concentration of SB431542 in the target cell reprogramming medium is approximately 1 μM to approximately 5 μM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, or approximately 5 μM. Preferably, the concentration of SB431542 is 3 μM.

[0118] In one embodiment, the concentration of ISX-9 in the target cell reprogramming medium is approximately 1 μM to approximately 20 μM, for example, approximately 1 μM, approximately 2.5 μM, approximately 5 μM, approximately 7.5 μM, approximately 10 μM, approximately 12.5 μM, approximately 15 μM, approximately 17.51 ​​μM, or approximately 20 μM. Preferably, the concentration of ISX-9 is 10 μM.

[0119] In one embodiment, the target cells are mesodermal-derived cells such as heart cells, ectodermal-derived cells such as nerve cells, and endoderm-derived cells such as colon cells. For example, the target cells can be neurons, skeletal muscle cells, hepatocytes, fibroblasts, osteoblasts, chondrocytes, adipocytes, endothelial cells, interstitial cells, smooth muscle cells, cardiomyocytes, nerve cells, hematopoietic cells, pancreatic islet cells, or virtually any cell in the body.

[0120] In one embodiment, the target cells are neurons, and the target cell (neuron) reprogramming medium comprises CHIR99021, Forskolin, 616452, and ISX9. In one embodiment, the target cell (neuron) reprogramming medium comprises 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, and 10 μM ISX9. In one embodiment, the target cell (neuron) reprogramming medium is Neurobasal medium supplemented with FBS, N2, B27, GlutaMAX, bFGF, and CHIR99021, 616452, Forskolin, and ISX-9. In one embodiment, the target cell (neuron) reprogramming medium is Neurobasal medium supplemented with 5% FBS, 1% N-2 supplement, 2% B27 supplement, 1% GlutaMAX, 25 ng / ml bFGF, and 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, and 10 μM ISX-9. In some embodiments, the target cell (neuron) reprogramming medium is also supplemented with antibiotics, such as 1% penicillin-streptomycin.

[0121] In one embodiment, the target cells are skeletal muscle cells, and the target cell (skeletal muscle cell) reprogramming medium comprises CHIR99021, Forskolin, 616452, and SB431542. In one embodiment, the target cell (skeletal muscle cell) reprogramming medium comprises 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, and 3 μM SB431542. In one embodiment, the target cell (skeletal muscle cell) reprogramming medium is DMEM / M199 medium supplemented with KSR, FBS, GlutaMAX, NEAA, and CHIR99021, 616452, Forskolin, and SB431542. In one embodiment, the target cell (skeletal muscle cell) reprogramming medium is a DMEM / M199 (4:1) medium supplemented with 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, and 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, and 3 μM SB431542. In some embodiments, the target cell (skeletal muscle cell) reprogramming medium is also supplemented with antibiotics, such as 1% penicillin-streptomycin.

[0122] In another aspect, the present invention provides a method for reprogramming a starting cell into a target cell, the method comprising the following steps:

[0123] (a) The starting cell population is cultured in the initiation reprogramming medium of the present invention for a period of time not more than 11 days, for example about 1 to about 11 days, preferably 4 days;

[0124] (b) Culturing the cell population obtained in step (a) in the target cell reprogramming medium of the present invention; and

[0125] (c) Optionally, harvest the target cell population obtained in step (b).

[0126] In some implementations, the target cell reprogramming medium is replaced with fresh medium every 4 days in step (b).

[0127] In some implementations, the culture in step (b) is carried out for approximately 8-16 days, such as about 8 days, about 12 days, or about 16 days.

[0128] In some embodiments, the method does not include the step of exposing the cell population to exogenous transcription factors. For example, the method does not include exposing the cell population to exogenous transcription factors related to cell reprogramming, such as Oct4, Sox2, Klf4, and C-Myc. That is, the method for reprogramming cells of the present invention is a chemically induced (using small molecule compounds) reprogramming method.

[0129] This invention also relates to the use of the small molecule mixtures, compositions, and culture media of this invention in cell reprogramming.

[0130] IV. Reagent Kit

[0131] The scope of this invention also includes kits comprising the small molecule mixtures, compositions, culture media, and / or cells of the invention, along with instructions for use. The kit may further comprise at least one additional reagent. Kits generally include a label indicating the intended use of the kit contents. Terminology labels include any written or documented material provided on or with the kit or otherwise accompanied by the kit. In one embodiment, the kit of the invention is used to carry out the methods of the invention. In some preferred embodiments, the kit comprises the initiation reprogramming culture medium and / or target cell reprogramming culture medium of the invention. Example

[0132] method

[0133] MEF separation

[0134] MEF medium: High glucose Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX, 1% non-essential amino acids (NEAA), 0.055 mM 2-mercaptoethanol and 1% penicillin-streptomycin.

[0135] Mouse embryonic fibroblasts (MEFs) were isolated from ICR mouse embryos. In short, after removing the head, limbs, and internal organs, the E13.5 embryos were minced with scissors and dissociated in trypsin-EDTA at 37°C for 10 minutes. MEF culture medium was added, and after centrifugation, the MEF cells were collected and cultured.

[0136] XEN-like cells generated from fibroblasts

[0137] XEN reprogramming medium (initiated reprogramming medium): KnockOut DMEM supplemented with 10% KnockOut serum replacement (KSR), 10% FBS, 1% GlutaMAX, 1% NEAA, 0.055 mM 2-mercaptoethanol, 1% penicillin-streptomycin (Invitrogen), 50 ng / ml basic fibroblast growth factor (bFGF), and a specified small molecule mixture such as VC6FAE (0.5 mM valproic acid, 20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin, 0.05 μM AM580, and 5 μM EPZ004777).

[0138] MEF cells were seeded at 20,000 cells per well in 12-well plates containing MEF medium. For XEN induction, the medium was changed to XEN reprogramming medium on the second day, and then changed every 4 days up to 12 days.

[0139] Inducing skeletal muscle cells from the induced cells

[0140] Skeletal muscle reprogramming medium: DMEM / M199 medium (4:1) supplemented with 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin (Invitrogen) and a small molecule mixture C6FS (20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin and 3 μM SB431542).

[0141] MEF cells were seeded at 20,000 cells per well in 12-well plates containing MEF medium. For skeletal muscle cell induction, the medium was changed to XEN reprogramming medium on day 0 and then to skeletal muscle reprogramming medium on day 4 to induce muscle cells for 8–12 days. The skeletal muscle reprogramming medium was changed every 4 days.

[0142] From the induced cells to induce neurons

[0143] Neuronal reprogramming medium: Neurobasal medium supplemented with 5% FBS, 1% N2, 2% B27 supplement, 1% GlutaMAX, 1% penicillin-streptomycin (Invitrogen), 25 ng / ml bFGF and a small molecule mixture C6FI (20 μM CHIR99021, 10 μM 616452, 50 μM Forskolin and 10 μM ISX-9).

[0144] MEF cells were seeded at 20,000 cells per well in 12-well plates containing MEF medium. For neuron induction, the medium was changed to XEN reprogramming medium on day 0 and then to neuron reprogramming medium on day 4 to induce neurons for 8–12 days. The neuron reprogramming medium was changed every 4 days.

[0145] Immunofluorescence

[0146] Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After washing twice with PBS, cells were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 3% donkey serum for 1 hour at room temperature. Cells were then incubated with primary antibody overnight at 4°C. After washing three times with PBS, cells were incubated with secondary antibody (Jackson Immuno Research) at 37°C for 1 hour. Cell nuclei were stained with DAPI (Roche Life Science) for 5 minutes.

[0147] The primary antibodies are the following specific antibodies: rabbit anti-SALL4 (Abcam, 1:500), goat anti-SOX17 (R&D, 1:500), goat anti-GATA4 (Santa Cruz, 1:300), goat anti-GATA6 (R&D, 1:200), mouse anti-Tubb3 (Biolegend, 1:300), rabbit anti-Synapsin 1 (Ancam, 1:500), rabbit anti-Map2 (Millipore, 1:200), rabbit anti-Neun (Millipore 1:500), mouse anti-myosin heavy chain (R&D, 1:300), mouse anti-MyoD (Thermo Fisher, 1:200), mouse anti-muscle protein (Thermo Fisher, 1:200), and mouse anti-α-actin (Sigma, 1:500), goat anti-Alb (Bethyl, 1:500), and goat anti-Hnf4a (Santa Cruz, 1:500). The secondary antibodies used were FITC-conjugated and TRITC-conjugated (Jackson ImmunoResearch, 1:200). The primary antibodies used were Santa Cruz (1:500) and TRITC-conjugated (1:300).

[0148] RT-qPCR

[0149] Total RNA was extracted using the EasyPure RNA Kit (TransGen Biotech) and reverse transcribed into cDNA using TransScript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech). Real-time PCR was performed using a 2×T5 Fast qPCR Mix (TSINGKE Biological Technology) on a Quantagne q225 system (KUBO technology).

[0150] Single-cell RNA-seq

[0151] Single cells were manually picked after digestion, lysed, and cDNA synthesized (Li et al., 2017; Gu et al., 2019). Single-cell cDNA was then amplified and fragmented as per published procedures (Li et al., 2017a; Gu et al., 2019). Sequencing libraries were constructed (NewEngland Biolabs) and sequenced using paired-end 150-bp reads on the Illumina HiSeq X-Ten platform (Novogene). Raw reads were first separated by cell barcoding, then trimmed and aligned to the mm9 mouse transcriptome, and deduplicated using UMI information as previously described (Trapnell et al., 2010).

[0152] Data processing and analysis

[0153] Gene expression levels were normalized to log2(FPKM+1) across all large RNAseq datasets. Day 20 and XEN data, primary neuron data, and skeletal muscle data were adapted from the GEO DataSet (GEO IDs: GSE73631, GSE68715, GSE111671, GSE85251). Genes that changed by more than a threshold (1.5) throughout the reprogramming process (days 0-20) were considered regulated. These genes were grouped into 10 clusters using the K-means method. For dropout experiments, gene expression under C6FAE conditions was first compared in MEF at days 4 and 8. Transcription factors that increased by at least 1.5 were identified as upregulated and retained for subsequent analysis. To analyze the optimized mixture used to induce neuronal and skeletal muscle cell expression, the inventors limited the gene list to a combined group of the top 100 genes whose expression was higher in neuronal cells than in MEF and whose expression was higher in skeletal muscle cells than in MEF, and then PCA was performed based on this gene list. Based on the gene load of each component, primary neurons and skeletal muscle cells are mapped to principal component analysis coordinates.

[0154] Single-cell data were purged to remove cells in which several spiked RNAs were detected, and then normalized using the R package RUVSeq to remove batch effects (Risso et al., 2014). Mean and dispersion were considered when acquiring variable genes for principal component analysis and t-distributed random neighborhood embeddings. A transcription factor gene correlation network was constructed by calculating the correlations between gene pairs, retaining only marginal values ​​with correlations less than -0.4 (negative correlation) or greater than 0.4 (positive regulation).

[0155] For the T-distributed random neighborhood embedding (van der Maaten, 2014), the inventors used Dmitry Ulyanov's multicore-tsne Python implementation. To accelerate computation, the inventors first used principal component analysis to reduce the dimensionality of the data, limiting it to the first 50 dimensions. The inventors used the default T-SNE parameters: perplexity = 30, learning rate = 200, early exaggeration = 12. The algorithm allowed 1500 iterations. To ensure that the T-SNE embedding faithfully represents the fundamental features of the cell state rather than batch effects, the inventors corrected the counting matrix used for batch effects using the Mutual Nearest Neighbor (MNN) algorithm (Haghverdi et al., 2018). The specific MNN implementation is mnnpy with parameters k = 15, sigma = 100, and a highly variable subset of genes, found using the Python framework Scanpy (Wolf et al., 2018), which executes Seurat's algorithm (Satija et al., 2015).

[0156] In order to cluster cells, such as Figure 6 As shown in Figure A, the inventors used the Louvain algorithm (Blondel et al., 2008), specifically Vincent Traag's open-source Python implementation. A resolution of 0.95 was used.

[0157] Reagent suppliers and product numbers

[0158] 1 KnockOut DMEM Thermo Fisher 10829018 2 KnockOut Serum Replacement(KSR) Thermo Fisher A3181502 3 Fetal Bovine Serum (FBS) VISTECH VIS146975 4 GlutaMAX Gibco 2059987 5 Non-Essential Amino Acid Gibco 2027433 6 penicillin-streptomycin Gibco 2029636 7 Neurobasal medium Thermo Fisher 21103049 8 N-2 Supplement Thermo Fisher A1370701 9 B27 supplement Thermo Fisher 17504044 10 CHIR99021 selleck S2924 11 Forskolin selleck S2449 12 616452 selleck S7223 13 AM580 MCE HY-10475 14 EPZ00477 selleck S7353 15 ISX-9 selleck S7914 16 Medium 199 Gibco 11150059 17 DMEM HyClone AE27369267 18 SB431542 selleck S1067 19 Recombinant Human FGF-4 PEPROTECH 100-31

[0159] The CAS number and structural formula of the compound used in this invention

[0160]

[0161]

[0162]

[0163] Example 1: Chemical mixtures activate endogenous expression of transcription factors from multiple lineages.

[0164] To investigate how chemical compounds determine cell fate, the inventors measured gene expression profiles by RNA sequencing during reprogramming of mouse embryonic fibroblasts (MEFs) into XEN-like cells at days 0, 4, 8, 12, and 20 of treatment with different chemical mixtures (consisting of CHIR99021, Forskolin, 616452, AM580, and EPZ00477; VPA, CHIR99021, Forskolin, 616452, AM580, and EPZ00477; or CHIR99021, Forskolin, 616452, EPZ00477, UNC0638, and CH55). Figure 1 A)

[0165] As shown in the figure, the dynamic display of variable patterns of gene expression induced by C6FAE ( Figure 1 B), consisting of 10 clusters, is divided into 4 groups in a stepwise manner ( Figure 1 B): Fibroblast genes downregulated in cluster 1 / 2 during the first 4 days, genes upregulated in clusters 3 / 4 / 5 from day 4 to day 8, XEN genes upregulated in clusters 6 / 7 / 8 / 9, and major fibroblast genes reduced in cluster 10 during the final period. Figure 1 C and 2A). Surprisingly, the genes upregulated during fibroblast reprogramming into XEN-like cells (clusters 3 / 4 / 5) included many lineage-associated transcription factors (TFs), such as Ascl1, Zic1, Hand2, Hey2, Ptf1a, and Gata3, which have been reported to regulate the development of the ectoderm, mesoderm, and endoderm, respectively. Figure 1 This gene expression pattern can also be detected in chemical reprogramming with other mixtures, even those containing additional chemical enhancers for XEN production (VPA, UNC0638, and CH55). Figure 1 E, 2B, and 2C). The highest-ranking related gene categories for these genes include "regulation of multicellular biological processes," "regulation of cell differentiation," and "forebrain development." Figure 1 F). Therefore, the inventors refer to this unique state as "chemically activated multi-lineage initiation" (CaMP).

[0166] To explore whether CaMP can only be generated from more immature developmental stages. In addition to induction in embryonic fibroblasts, the inventors also induced CaMP using mouse neonatal dermal fibroblasts (NDF) and adult mouse tail tip fibroblasts (TTF). Genome profiling showed that the reprogramming mixture also activated many developmentally relevant TFs in these cells, which possessed similar but slightly different characteristics. Figure 2 (D and 2E).

[0167] The XEN master gene was significantly activated in the final stage, indicating that XEN cell fate is induced in a stepwise manner rather than determined in the initial stages of chemical reprogramming.

[0168] CHIR99021 is a GSK-3α / β inhibitor; 616452 is a TGFβR-1 / ALK5 inhibitor; Forskolin is a cAMP activator; AM580 is a RARα activator; and EPZ004777 is a DOT1L inhibitor. Furthermore, UNC0638 is a selective inhibitor of protein lysine methyltransferases (HMTases) such as G9a and GLP, and CH55 is an activator of RAR-α and RAR-β. It is anticipated that combinations of other compounds exhibiting the same inhibitory / activating activity in the same signaling pathway can also achieve the "chemically activated multi-lineage initiation" of this invention.

[0169] Example 2: By fine-tuning the composition of chemical substances, CaMP cells were specialized into cell types of different germ layers, serving as seeds.

[0170] Since the CaMP step may have already prepared cells for multi-lineage specialization, these cells have endogenous expression of cell type-related major genes. The inventors then investigated whether other cell types (e.g., neurons, muscle cells) could be induced by altering the culture medium and / or chemical mixture after CaMP induction.

[0171] To specialize neurons, the inventors modified the neuron-preferred culture medium and attempted to fine-tune the mixture after the CaMP process. The inventors discovered that the mixture C6FI (CHIR99021, 616452, Forskolin, and ISX9) significantly promoted the conversion from CaMP to neuron-like cells. Figure 4 A and 4B). The induced neuron-like cells obtained expressed neuronal markers Tuj1, Map2, Syn1, and NeuN, and typical neuronal morphology was detected by immunostaining. Figure 3 A). Furthermore, these induced neuron-like cells were also detected by quantitative PCR to express a set of functional markers: Rbfox3, Gabbr2, and Chat( Figure 3 B). Importantly, almost no neurons were induced 12 days after using C6FI alone, unless the cells underwent the CaMP phase (…). Figure 3(C) This indicates that the CaMP state induced by brief treatment with XEN reprogramming medium provides significantly enhanced plasticity for chemical reprogramming. To determine whether an intermediate XEN-like state (which is also reported to be plastic) was induced, the inventors knocked out the XEN major genes Sall4 and Gata4 during neuron-like cell induction. The inventors found that their knockout significantly reduced XEN colony formation without reducing the efficiency of neuron-like cell generation. Figure 4 The presence of C, 4G, and 4H indicates that CaMP cells already possess plasticity independent of XEN-like cell generation.

[0172] Furthermore, by switching to a myocyte culture medium containing compounds CHIR99021, 616452, Forskolin, and SB431542 (C6FS) in the second step, the inventors very effectively induced skeletal muscle cells expressing MyHC, Myog, Myod1, and Tnnt3. Figure 3 D and 3E). Just like neuronal reprogramming, effective skeletal muscle reprogramming also requires CaMP induction (D and 3E). Figure 3 F). The inventors optimized the mixture for the CaMP phase through dropout experiments and found that subtracting CHIR99021 resulted in the highest efficiency for skeletal muscle cell induction. Figure 4 D). Compounds CHIR99021, Forskolin, 616452, and SB431542 are all essential for skeletal muscle induction. Figure 4 E). Knockdown of the XEN major gene does not impair neuronal induction efficiency, suggesting the existence of a more direct molecular pathway bypassing the XEN-like state. Figure 4 F, 4G, and 4H).

[0173] Through RNA sequencing, the inventors discovered that CaMP-induced activation of neuron-specific and muscle cell-specific genes in neurons and muscle cells, respectively (…). Figure 3 G, 3H). Through principal component analysis, the inventors discovered that neurons and muscle cells induced by CaMP-C6FI and CaMP-C6FS have a transcriptional state closer to that of primary functional cells than cells without induction. Figure 3 I).

[0174] In summary, the inventors were able to effectively induce cell types from different germ layers from CaMP cells, such as skeletal muscle cells and neurons. Figure 3 J). Initiating multiple lineages through simple compound manipulation may offer excellent new opportunities for obtaining other lineages.

[0175] Example 3: CaMP is characterized by the random expression of endogenous development-related transcription factors.

[0176] To investigate whether unexpectedly upregulated multidevelopmental genes are activated simultaneously or separately in each single cell at different expression levels, the inventors further studied the reprogramming process using single-cell RNA sequencing. During the C6FAE-mediated MEFXEN reprogramming process, the inventors sampled 808 single cells at 10 time points and used the highly sensitive single-cell RNA sequencing method SMART-seq2 to assess the expression of genome-wide genes (including TFs with low transcriptional levels in single cells). Figure 6 A). After extensive data verification and correction for technical noise and batch effects ( Figure 6 (B, 6C, and 6D), 610 cells were retained for further analysis.

[0177] In single cells, the mRNA levels of many lineage-specific genes were upregulated within the first 5 days. Figure 5 A, 5B, and 6E), which is consistent with the inventors' extensive RNA sequencing data. Of the altered endogenous TFs, 78.96% (using two-sided Wilcoxon rank-sum test, total 442, p < 0.05) were upregulated, while only a small number were suppressed 5 days after induction. Figure 5 C).

[0178] Interestingly, these genes do not exhibit any carefully designed dynamic patterns in every cell, but are activated randomly. The activated genes are hardly related to each other. Figure 5 (D and 5E). Cells have different numbers of activating genes, and these genes are upregulated at different time points from day 1 to day 5. Figure 6 F).

[0179] Due to the low expression of XEN TF in activated cells and the high expression of the major gene in fibroblasts, the chemical mixture randomly activates the endogenous expression of TF from multiple lineages rather than preferentially considering XEN cell fate. Figure 5 F). When its major genes, such as Sox17 and Sall4, are highly expressed ( Figure 6 G) and when most TFs of other lineages are downregulated, the fate of XEN cells emerges from this multi-lineage initiation state, which further suggests a two-step model (multi-lineage initiation and specialization) during XEN-like cell fate reprogramming.

[0180] Example 4: Sox17 activates progressive cell fate specialization and conversion in cells.

[0181] Since it is unclear how non-cell-type-specific plastic CaMP states are driven to specific cell types, the inventors next explored how XEN-like cell fates emerge from populations in the early stages of chemical reprogramming to become CiPSCs. The inventors employed principal component analysis (PCA) and t-distributed random neighborhood embedding (t-SNE) analysis of single-cell RNA-seq data. These two well-tested methods yielded similar profiles that captured the overall trend of cellular changes from day 0 to day 20. Figure 5 (A and 6E). Cells were initially very close to each other, then gradually separated, eventually branching into two branches. These two branches consisted primarily of cells from day 12 and day 20, and they belonged to two of eight clusters in which cells were grouped together using Louvain clustering at a resolution of 0.95 (Blondel et al., 2008; Levine et al., 2015; Wolf et al., 2018). Figure 8 A and 8B).

[0182] Cells located in the left branch of the t-SNE diagram (cluster 8) exhibit significant expression levels of the major XEN genes Sox17, Sall4, and Gata4, indicating that they are successfully induced XEN-like cells. The other branch, the trapped state, includes cells with lower expression of Sall4 and Gata4, which still retain fibroblast gene expression, suggesting that they failed to acquire a new cell fate. Figure 5 F).

[0183] To more broadly evaluate cell fate in two distinct branches, the inventors used extensive RNA-seq data from the top 1000 differentially expressed genes MEF and XEN to assess cell identity using a secondary programming-based analysis technique (Treutlein et al., 2016). Fibroblast identity moderately decreased in the early stages and remained constant until XEN identity emerged, while XEN identity slowly upregulated in the first 8 days, with most improvement occurring after day 8. Figure 8 C). The transition from MEF cell fate to XEN cell fate was very rapid within the last 4 days. Consistent with the marker gene, the left branch exhibited the highest XEN isotype and the lowest MEF isotype. Figure 7 A) indicates that XEN reprogramming has been completed, which the inventors refer to as the "successful branch." The right branch, due to the highest MEF identity, is called the "trapped branch." Interestingly, these two cell fates never showed high scores in the same single cell, implying that they are competing and cannot coexist in the same cell.

[0184] Next, the inventors inquired whether the trapped branches could be reprogrammed into XEN cells. To answer this question, the inventors selected a single clone with weak Epcam expression and the ambiguous morphology of its trapped branches. The colony was digested into single cells using trypsin and seeded into isolated wells. Figure 8 D). Even after culturing for another 12 days in C6FAE medium, single cells from the captured community rarely formed XEN communities. Figure 8 E). This result indicates that cells in trapped branches cannot be converted into successful branches and reprogrammed into XEN cells.

[0185] The biggest difference between the successful and trapped branches is the differential expression of XEN and the major fibroblast gene in the latter. Figure 7 B). XEN major TFs, especially Sox17, have the highest expression in successful branches. The expression order of XEN major TFs is Sox17, Sal4, Gata4, and then Foxa2. Figure 8 F), indicating that Sox17 is upstream of other XEN genes. Through immunostaining, the inventors found that cells expressing Sall4, Gata4, and Foxa2 were all contained within the Sox17-expressing community. Figure 7 C). Knocking out Sox17 from the start of reprogramming inhibits XEN formation and severely damages other major XEN genes. Figure 7 D). Furthermore, overexpression of Sox17 promotes the upregulation of Gata4 and Foxa2 (D). Figure 8 G). These findings further confirm the view that Sall4, Gata4, and Foxa2 emerge after Sox17, and that Sox17 is the top-level upstream gene of the XEN master gene in the hierarchical structure of the gene regulatory network.

[0186] Importantly, the activation level of Sox17 during the CaMP process determines cell fate specialization and transition. A mathematical model of hierarchical regulation among the XEN major genes reveals a saddle-shaped branching in the gene expression dynamics system, with two stable fixations and one unstable fixation point. Figure 7 E). Following activation of Sox17 in the CaMP step, the cell state moves to a near-unstable fixation point, then transitions to either of two stable fixation points, representing successful branching and trapped branching, respectively. Higher Sox17 expression in the CaMP step transfers cells to a stable fixation point with higher Sall4 and Gata4 expression levels. Figure 7 E and 8H), thus completing the core regulatory network for cell fate specialization and conversion ( Figure 7F). In the XEN subnetwork, Sox17 is the major regulator that interacts most extensively with other genes, including fibroblast major genes. Overexpression of Sox17 promotes the downregulation of fibroblast core genes. Figure 7 G), conversely, downregulation of fibroblast core genes promotes upregulation of elements in the XEN core regulatory network (G). Figure 7 Sox17 (H and 8I) is therefore one of the most important TFs activated during the CaMP process, defining cell fate specialization and conversion.

[0187] Example 5: Chemical enhancers are beneficial for different stages of reprogramming.

[0188] Although the same mixture can be used for both plasticization and specialization, the uncovered two-step "plasticization and specialization" process raises the possibility that different chemical mixtures are required for different steps. To verify this possibility, the inventors first compared the gene expression profiles of C6FAE-induced cells with and without chemical enhancers at different steps. The results showed that CH55 promoted the expression of Sox17 and Sall4 in the first 4 days, while inhibiting further expression of the XEN gene from day 4 to day 12. Figure 9 (A and 9B). This indicates that CH55 functions only in the first step by promoting the expression of Sox17, one of the early initiating genes in the CaMP step.

[0189] However, another chemical enhancer, valproic acid (VPA), improved reprogramming efficiency in a different way. It promoted the upregulation of the XEN gene over the last 8 days. Figure 9 A), while starting from day 4, other lineage-specific genes are suppressed ( Figure 9 B). However, VPA down-registered the Sox17 expression on step 4. This indicates that VPA only improved XEN reprogramming in step 2 by supporting up-regulation of the XEN network.

[0190] Similarly, through immunofluorescence, the inventors found that the VC6FAE mixture primarily induced "smooth" communities co-expressing Sox17, Gata4, and Sall4, while the C6FAE mixture induced many "fuzzy" communities with strong Sox17 expression and impaired Sall4 and Gata4 expression. Figure 10 A, 10B, and 10C). The fuzzy communities exhibited higher transcriptional levels of the fibroblast major genes Osr1, Prrx1, and Twist2. Figure 10 D). These findings suggest that VPA improves C6FAE-mediated XEN reprogramming by promoting XEN specialization and suppressing genes specific to other lineages.

[0191] The inventors further determined whether XEN reprogramming could be further promoted by stepwise use of chemical enhancers, specifically by adding CH55 and VPA at different steps. The inventors found that adding CH55 in the first 4 days promoted the expression of Sox17 and Sal4 more strongly than adding CH55 throughout the entire reprogramming process. Figure 9 C and 10E). Compared to adding VPA throughout the reprogramming process, adding VPA within the last 8 days promoted more Gata4 and Foxa2 transcription (C and 10E). Figure 9 D and 10F). Importantly, sequential use of CH55 and VPA in different steps yields the highest reprogramming efficiency to produce XEN-like communities (D and 10F). Figure 10 (G and 9E). These data further support the two-step reprogramming hypothesis, and the specified chemical synergists can facilitate reprogramming at different steps, and may have different mechanisms.

[0192] Example 6: The core reprogramming chemical (FC6) coordinates the CaMP process and the specialization process through different mechanisms.

[0193] The inventors further inquired about the role of the core reprogramming chemicals CHIR99021, Forskolin, and 616452 (C6F) in the biphasic "plasticization and specialization" process. Numerous RNA expression profiles were analyzed from samples treated with the mixture, which subtracted each of the three chemicals at different stages.

[0194] The inventors discovered that CHIR99021, 616452, and Forskolin are essential for the activation of most lineage-specific TFs. Among genes activated by the C6FAE mixture, 44 and 48 genes, respectively, failed to activate on day 4 and day 8 without any of the three compounds. Figure 11 A and 11B). The subtraction of CHIR99021, 616452, or Forskolin respectively inhibited a small portion of TF expression ( Figure 11 C and 11D). Furthermore, a larger proportion of TF was induced by CHIR99021, Forskolin, and 616452 together, because subtracting any one of these compounds hindered their activation. Figure 11 C and 11D). Importantly, one of the chemically induced genes, Sox17, also requires all three core compounds to activate its expression. Subtracting any one of CHIR / FSK / 616 from day 0 inhibits the expression of the major XEN genes, especially Sox17 (C and 11D). Figure 11 (C, 12A, and 12B). Therefore, the inventors conclude that CHIR99021 / Forskolin / 616452 jointly contribute to the CaMP process.

[0195] However, CHIR99021 / Forskolin / 616452 plays a different role in the specialization process. Since Sal4 and Gata4 are core TTFs in the XEN cell fate core regulatory network, the inventors analyzed the effects of CHIR99021 / Forskolin / 616452 on Sal4 and Gata4 expression. By subtracting each of them from day 4 to day 12, the inventors found that the mixture containing CHIR99021 and Forskolin promoted Gata4 expression at both the mRNA and protein levels. Figure 11 E, 11F, and 11G indicate that these two compounds function through Gata4 activation. Furthermore, subtracting 616452 blocked Sall4 upregulation, and all mixtures containing 616452 upregulated both Sall4 mRNA and protein levels. Figure 11 E, 11H, and 11I) indicate that 616452 plays a crucial role in Sall4 activation. This is because Sall4 expression promotes the upregulation of Gata4 (E, 11H, and 11I). Figure 12 In addition to C, 12D, and 12E, the inventors can also detect Gata4 expression in Sall4-positive communities. In summary, CHIR99021 / Forskolin / 616452 simultaneously induces multi-lineage TFs, including Sox17, in the CaMP step, while CHIR99021 / Forskolin and 616452 activate Gata4 and Sall4, respectively, during the specialization phase, thus establishing the entire core regulatory network of XEN. Figure 11 J). After day 8, CHEN99021 / Forskolin / 616452 was not required for XEN gene expression. Figure 12 F) indicates that the transition period is a self-organizing process of the XEN master gene regulatory network. The different functions of CHIR99021 / Forskolin / 616452 in the two stages further support the hypothesis that XEN reprogramming can be considered a stepwise process.

[0196] In this invention, the inventors provide a novel method for generating functional cells in regenerative medicine by inducing a "chemically activated multilineage initiation (CaMP)" state through simple chemical treatment. This strategy may have some major advantages over the iPS strategy, namely, bypassing the long-term culture required for iPS cell induction. Furthermore, this strategy may be more direct and easier to operate. Additionally, cell types from different germ layers, such as skeletal muscle cells, neurons, and extraembryonic XEN-like cells, can be obtained by inducing CaMP cells. Figure 13 This potential for differentiation only exists in childhood. It has been described in the context of pluripotency.

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Claims

1. A method for reprogramming starter cells into target cells, the method comprising a first phase and a second phase, The first stage includes exposing the starting cell population to a mixture of small molecules that induce reprogramming for a period of time to obtain an induced cell population, wherein the period of time is no more than 11 days; The second stage involves exposing the induced cell population obtained in the first stage to a target cell reprogramming small molecule mixture, thereby obtaining the target cell population. The initiating cells are mouse fibroblasts, the target cells are neurons or skeletal muscle cells, and the reprogramming-inducing small molecule mixture consists of CHIR99021, Forskolin, 616452, AM580, and EPZ00477. When the target cell is a neuronal cell, the target cell reprogramming small molecule mixture consists of CHIR99021, Forskolin, 616452 and ISX9; or when the target cell is a skeletal muscle cell, the target cell reprogramming small molecule mixture consists of CHIR99021, Forskolin, 616452 and SB431542.

2. The method of claim 1, wherein the time period of the first stage is 1-11 days.

3. The method of claim 1, wherein the time period of the first stage is 4 days.

Images