In vitro induction of adult stem cell expansion and induction

Abstract

The present invention generally relates to compositions and methods for inducing the expansion and / or induction of adult stem cells (ASCs) in vitro using miR-302-like pre-miRNAs, shRNAs and / or siRNAs containing the shared sequence 5'-UAAGUGCUUC CAUGUUU-3' at their 5'-ends, in combination with certain wound healing-related specific factors, including, but not limited to, basic fibroblast growth factor (bFGF) / fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), stem cell factor (SCF), homeobox protein (HOX), Notch, GSK, Wnt / beta-catenin signaling, interleukins, and / or bone morphogenetic proteins (BMPs). The principles of the present invention relate to a novel mechanism of inducible symmetric ASC division recently discovered in an in vivo skin wound healing model, resulting in expanded ASCs useful for the treatment of a variety of aging- and cellular injury-related human diseases, including, but not limited to, Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, AIDS, leukemia, lymphoma, many types of cancer, and aging.

Description

[Technical Field]

[0001] Priority: The present invention claims priority to U.S. Provisional Application No. 62 / 692,862, filed July 2, 2018, entitled "In Vitro Induction of Expansion of CD34-Positive Adult Stem Cells," the entire contents of which are incorporated herein by reference.

[0002] Field of the invention: The present invention generally relates to compositions and methods for inducing the expansion and / or induction of adult stem cells (ASCs) in vitro using small non-coding RNAs (snRNAs) in combination with certain specific protein factors, for example, using small hairpin RNAs (shRNAs), microRNA precursors (pre-miRNAs) and / or short interfering RNAs (siRNAs) in combination with one or more specific factors consisting of basic fibroblast growth factor (bFGF) / fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), stem cell factor (SCF), homeobox protein (HOX), Notch, GSK, Wnt / β-catenin signaling, interleukins, and / or bone morphogenetic proteins (BMPs). In particular, the present invention provides a method for the production of CD34-positive adult stem cells (CD34) using miR-302-mimicking shRNA, pre-miRNA and / or siRNA containing a shared sequence homologous to 5'-UAAGUGCUUC CAUGUUU-3', in combination with one or more specific factors selected from bFGF / FGF-2, LIF, IGF, EGF, PDGF, VEGF, TGF, TNF, SCF, HOX, Notch, GSK, Wnt / β-catenin, interleukins and / or BMPs. +The present invention relates to compositions for inducing the expansion and / or induction of ASCs (autosomal dominant stem cells) in vitro and methods for using the same. The principle of induced ASC expansion and induction is related to a novel mechanism of induced symmetric division of adult stem cells recently discovered in an in vivo skin wound healing model. The resulting expanded ASCs are useful for treating various aging- and cell damage-related diseases, including, but not limited to, Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, AIDS, leukemia, lymphoma, and many types of cancer. In particular, the present invention is useful for preventing aging and treating aging-related degenerative diseases by providing the body with unlimitedly expanded ASCs to repair and revitalize damaged and / or aging tissues and organs in vivo. [Background technology]

[0003] Stem cells are a treasure chest of life containing numerous active ingredients that can help promote the regeneration of new cells / tissues, repair and / or revitalize damaged / aged tissues, treat degenerative diseases, and prevent the development and progression of tumors / cancer. Therefore, these stem cells can be used as tools for novel drug screening, identification, isolation, and production. Consequently, the resulting drugs can be useful for developing pharmaceutical and therapeutic applications, such as biomedical uses, research devices and / or equipment, diagnostics and / or treatments, and combinations thereof.

[0004] MicroRNAs (miRNAs) are one of the main active components in human embryonic stem cells (hESCs). Major hESC-specific miRNAs include, but are not limited to, miR-200, miR-290–295, miR-302, miR-371–373, and miR-520 families. Among them, the miR-302 family has been found to play an important role in maintaining pluripotency and tumor suppression (Lin and Ying, 2008; Lin et al., 2008, 2010, and 2011). MiR-302 contains eight family members, including four sense miR-302 (a, b, c, d) and four antisense miR-302* (a*, b*, c*, d*) sequences. These sense and antisense members are partially complementary and can form double-helix structures. For example, the precursors of miR-302 are hairpin-shaped small RNAs formed by duplexes (stem arms) of miR-302a and a* (pre-miR-302a; SEQ.ID.NO.1), miR-302b and b* (pre-miR-302b; SEQ.ID.NO.2), miR-302c and c* (miR-302c; SEQ.ID.NO.3), and miR-302d and d* (pre-miR-302d; SEQ.ID.NO.4), respectively, with a base sequence linked to one end (stem loop) of the duplex. To activate miR-302 function, the precursor of miR-302 (pre-miR-302) is first processed by cellular RNase III Dicer to mature miR-302 and miR-302*, which then form an RNA-induced silencing complex (RISC) with certain Argonaute (AGO) proteins, leading to either RNA interference (RNAi)-directed degradation or translational repression of target gene transcripts (mRNAs), especially those of developmental and oncogenes (Lin et al., 2008, 2010, 2011).

[0005] MiR-302 is the most abundant non-coding RNA (ncRNA) species in human ES cells (hESCs) and induced pluripotent stem cells (iPSCs). Our previous studies have shown that ectopic overexpression of miR-302 above levels found in hESCs can reprogram human normal and cancer tissue cells into hESC-like iPSCs with a relatively slow cell cycle rate (20–24 h / cycle) similar to that of early human fertilized eggs at the morula stage (Lin et al., 2008, 2010, and 2011; Lin et al., EP 2198025, US 9,567,591, US 9,394,538, and US Patent Application No. 12 / 318,806). Although relative quiescence is a specific property of these miR-302-induced iPSCs, hESCs and other previously reported four-factor-induced (Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28) iPSCs exhibit proliferative cell cycle rates (12–15 h / cycle) similar to those of tumor / cancer cells (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007; Wang et al., 2008). To investigate the tumor-suppressive effects of miR-302, we confirmed the involvement of two miR-302-targeted G1-checkpoint regulators, including cyclin-dependent kinase 2 (CDK2) and cyclin D (Lin et al., 2010; Lin, US Patent Nos. 9,394,538 and 9,422,559). Progression through the cell cycle is known to be driven by the activation of cyclin-dependent kinases (CDKs), which form functional complexes with negative regulators, CDK inhibitors, as well as positive regulatory subunits, cyclins (CKIs, e.g., p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1, p27Kip1, etc.). In mammals, different cyclin-CDK complexes are involved in regulating different cell cycle transitions, such as cyclin-D-CDK4 / 6 for G1 phase progression, cyclin-E-CDK2 for G1-to-S transition, cyclin-A-CDK2 for S phase progression, and cyclin-A / B-CDC2 (cyclin-A / B-CDK1) for entry into M phase.As a result, our study demonstrated that the tumor-suppressive function of miR-302 stems from co-inhibition of the cyclin-E-CDK2 and cyclin-D-CDK4 / 6 pathways at the G1 to S transition.

[0006] Although miR-302 is useful for the design and development of novel anticancer drugs and vaccines, its productivity is problematic because natural miR-302 and its precursor are found only in human pluripotent stem cells, particularly hESCs, making their availability extremely limited. Alternatively, synthetic small interfering RNA (siRNA) can be used to mimic the natural miR-302 precursor (pre-miR-302). However, because the stem-arm region of the hairpin-like pre-miR-302 structure is formed from two imperfectly complementary strands, sense miR-302 and antisense miR-302*, perfectly matched synthetic siRNA-302 mimics cannot replace the function of natural miR-302*, whose sequence differs from the antisense strand of the siRNA. For example, the antisense strand of the siRNA-302a mimic is 5'-UCACCAAAAC AUGGAAGCAC UUA-3' (SEQ ID NO. 5), while the natural miR-302a* is 5'-ACUUAAACGU GGAUGUACUU GCU-3' (SEQ ID NO. 6). Because the full function of miR-302 derives from both its sense and antisense miR-302* strands, previous studies using these siRNA mimics have often shown results that differ from the function of the actual natural miR-302. Furthermore, due to the high degree of nucleotide mismatch (23%-46%) between the sense and antisense miR-302* sequences, synthetic miR-302 and miR-302* are unlikely to spontaneously form the correct duplex structure, especially without the aid of a stem-loop structure. The stem-loop structure of pre-miRNAs functions by ensuring that the two mismatched sense and antisense miRNA sequences are sufficiently close and long to form a stable, correct duplex. Meanwhile, our recent findings on iPSCs may provide an alternative source for the production of pre-miR-302 (Lin, EP 2198025, US 9,567,591, and US Patent Application No. 12 / 318,806). However, the cost of growing iPSCs remains too high for industrial production.

[0007] In addition to generating iPSCs, our recent studies further demonstrated that miR-302 promotes the generation of CD34-positive adult stem cells (CD34+Lin has discovered that it can also induce the expansion and induction of adult stem cells (ASCs) (US Pat. No. 9,879,263 and US Patent Application No. 15 / 661,346). Prior art attempts to expand adult stem cell (ASC) populations include Moon's US Pat. No. 7,850,960, which uses GSK-3; Moon's US Pat. No. 8,372,397 and European Patent No. EP2415480A2, which uses the Wnt / β-catenin signaling pathway; Rudd's US Pat. No. 11 / 614,345, which uses G-CSF / GM-CSF and SCF; and Rudnicki's US Pat. No. 13 / 266,428, which uses Wnt7a. In addition, several scientific reports have demonstrated the involvement of HOXB4 and Notch1 / 4 in inducing the expansion of human CD34-positive hematopoietic stem cells (HSCs) in vitro (Antonchuk et al., Cell 109: 39-45, 2002; Karlsson S., Blood 104: 2210-2211, 2004; Schiedlmeier et al., PNAS USA 104: 16952-16957, 2007). Miller et al. (PNAS USA 94: 13648-13653, 1997) further demonstrated the use of IL-6 / 11, flt3-ligand, and Steel factor (SF) to induce the expansion of murine (CD34-negative) HSCs in vitro. However, as noted in a recent review by Walasek et al. (Ann. NY Acad. Sci. 1266: 138-150, 2012), "Attempts to improve hematopoietic reconstitution and transplantability of ex vivo expanded hematopoietic stem and progenitor cells (HSPCs) have met with little success due to the inability to generate sufficient stem cell mass and excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) expand rapidly after transplantation in vivo, experience from in vitro studies indicates that controlling the self-renewal and differentiation of HSPCs in culture remains difficult. Protocols based on hematopoietic cytokines cannot support reliable expansion of immature stem cells in culture, indicating the need for additional factors." It should be noted, in particular, that expanded ASCs cannot be repeatedly cultured in vitro or ex vivo for multiple passages.As a result, all prior art techniques can only provide a maximum amplification of the starting ASC population by tens to hundreds of times, which is not sufficient for clinical therapeutic applications.

[0008] Given the low amplification rate and poor pluripotency of conventional ASC expansion methods, new solutions that overcome these problems are desirable. Clearly, as Walasek states, additional critical factors are required to achieve this goal, but no one knows what they are. Therefore, in our recent research, we have identified a new critical factor that overcomes all of the problems of the prior art by using a small microRNA (miRNA), miR-302, rather than a protein. Prior art techniques overlooked miRNAs due to their unknown function in DNA demethylation, which is necessary to unlock natural barriers in the cellular genome, such as gene methylation and histone acetylation, thereby enabling the full mechanism of gene regulatory steps necessary to induce ASC expansion and / or induction under various in vitro, ex vivo, and in vivo conditions. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent Application Publication No. 2198025 [Patent Document 2] U.S. Patent No. 9,567,591 [Patent Document 3] U.S. Patent No. 9,394,538 [Patent Document 4] U.S. Patent No. 9,422,559 [Patent Document 5] U.S. Patent No. 9,879,263 [Patent Document 6] U.S. Patent No. 7,850,960 [Patent Document 7] U.S. Patent No. 8,372,397 [Patent Document 8] European Patent Application Publication No. 2415480 [Non-patent literature]

[0010] [Non-Patent Document 1] Lin SL and Ying SY. (2008) Role of mir-302 microRNA family in stem cell pluripotency and renewal. Ying SY. (Ed.) Current Perspectives inMicroRNAs. Springer Publishers press, New York, pp 167-185 [Non-patent document 2] Lin SL, Chang D, Chang-Lin S, Lin CH, Wu DTS, Chen DT, and Ying SY. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115-2124 [Non-patent document 3] Lin SL, Chang D, Ying SY, Leu D, and Wu DTS. (2010) MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4 / 6 cell cycle pathways. Cancer Res. 70, 9473-9482 [Non-patent document 4] Lin SL, Chang D, Lin CH Ying SY, Leu D, and Wu DTS. (2011) Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res. 39, 1054-1065 [Non-patent document 5] Takahashi et al. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676

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[0011] The principle of the present invention is primarily based on the DNA demethylation function of miR-302. It is conceivable to use other small RNA molecules, such as shRNA, siRNA, and hairpin-shaped pre-miRNA, or protein factors and chemicals with similar functions to replace miR-302 for the same purpose of inducing DNA demethylation in cells and enabling ASC expansion and / or induction in vitro, ex vivo, and in vivo. It is well known that stem cells contain highly demethylated genomes to preserve and maintain pluripotency (i.e., hESCs and iPSCs) or pluripotency (i.e., ASCs). In the genome of a cell, DNA methylation acts as a lock to set the expression profile of all cell-type-specific genes, thereby prohibiting all possible reverse development or reprogramming of somatic cells back to stem cells. Consequently, DNA demethylation is the most important step required to unlock the genome and reset the gene expression pattern to a unique stem cell-specific profile to induce and maintain pluripotency or pluripotency in stem cell renewal and / or reprogramming. To study this DNA demethylation process, we first discovered the mechanism of miR-302-mediated DNA demethylation in hESCs and iPSCs (Lin and Ying, 2008; Lin et al., 2008 and 2011; Ying et al., 2018). Our previous studies have revealed that miR-302 downregulates multiple key epigenetic regulators, such as MECP1 / 2, AOF1 / 2 (also known as KDM1b / 1a or LSD2 / 1), DNMT1, HDAC2 / 4, and MBD2, facilitating genomic DNA demethylation and iPSC reprogramming (Lin and Ying, 2008; Lin et al., 2008, 2011, and 2013). For the same reason, miR-302 may be able to induce and maintain ASC regeneration and expansion by downregulating all or some of these key epigenetic regulators.

[0012] The strength of microRNA (miRNA)-mediated gene regulation depends on the concentration of the miRNA and the affinity between the miRNA and its target gene. In other words, a strong target gene exhibits higher affinity for the miRNA, and therefore requires a lower miRNA concentration to be suppressed, and vice versa. As a result, different target genes can be down-regulated using different levels of miRNA concentration. For example, while it has been discovered that somatic cells can be reprogrammed to form hESC-like iPSCs by using higher concentrations of miR-302 (higher than the miR-302 levels in hESC H1 and H9 lines) (Lin et al., 2008, 2010, and 2011; Lin, EP 2198025, US 9,567,591, US 9,394,538, and US Patent Application No. 12 / 318,806), our recent invention also demonstrates that the use of relatively low concentrations of miR-302 (approximately 10%-50% of the miR-302 levels in hESC H1 and H9) can induce the reprogramming of CD34 in vivo. + (Lin, US 9,879,263 and US Patent Application No. 15 / 661,346) This provides evidence that the mechanisms of these two distinct phenomena are likely distinct and may involve slightly different sets of genes targeted by miR-302. In particular, the use of miR-302 alone is not sufficient to induce CD34 expression in in vivo wound healing conditions, but rather in in vitro cell culture. +Because miR-302 can only induce ASC expansion, other specific factors involved in wound healing may need to work with miR-302 to set the ASC-specific gene profile necessary to induce ASC expansion and / or induction in vitro. Specific factors related to wound healing include, but are not limited to, basic fibroblast growth factor (bFGF) / fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), stem cell factor (SCF), homeobox protein (HOX), Notch, GSK, Wnt / β-catenin pathway signaling, interleukins, and / or bone morphogenetic proteins (BMPs).

[0013] The miR-302 family is the most abundant miRNA species in hESCs and iPSCs. All sense miR-302 family members share a consensus sequence identical to the 17 nucleotides at their 5' ends, i.e., 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID.NO.7), and the full-length 23-nucleotide mature sequence is over 82% identical. Based on the results of analyses using the online computational programs TARGETSCAN (http: / / www.targetscan.org / ) and PICTAR-VERT (http: / / pictar.mdc-berlin.de / ), these sense miR-302 members include miR-302a (SEQ ID NO. 8), miR-302b (SEQ ID NO. 9), miR-302c (SEQ ID NO. 10), and miR-302d (SEQ ID NO. 11), all of which can simultaneously target approximately 98% of the same genes, including over 600 human genes. Furthermore, miR-302 shares many overlapping target genes with the miR-92, miR-93, miR-200, miR-367, miR-371, miR-372, miR-373, miR-374, and miR-520 family members, which have similar functions. Most of its target genes are developmental signals and / or transcription factors involved in the initiation or establishment of lineage-specific differentiation during early embryogenesis (Lin et al., 2008 and 2011; Ying et al., 2018). Furthermore, because many of its target genes are oncogenes, miR-302 and its associated miRNAs may further function as tumor suppressors to prevent the misdirection of normal stem cells from regenerating or expanding into tumor / cancer cell development.

[0014] Stem cell research holds the key to developing novel regenerative medicines. Therefore, the present inventors focus their research on the screening, discovery, and isolation of novel drugs from stem cells to treat various human diseases. The emergence of iPSCs has significantly promoted progress in this research direction. Using iPSCs, the present inventors isolated and identified "glycylglycerin" as a novel sugar-like chemical group that protects embryonic stem cell-specific miRNAs (ESC-miRNAs), including miR-302, from degradation and maintains the pluripotency of iPSCs and ESCs (Lin, U.S. Patent No. 9,387,251). Our recent study (Lin SL, 2018) further demonstrated that some isolated glycylglycerin-bound ESC-miRNAs, particularly miR-302, can be used to induce CD34-positive adult stem cells (CD34) in vivo. + We have demonstrated that intact (complete) wound healing can be enhanced by inducing the expansion of ASCs (autosomal dominant cell). Since no similar mechanism has been reported previously, the present invention provides (1) a novel function of miR-302 and other wound healing-related specific factors involved in the mechanism of ASC expansion in vivo and in vitro, and (2) a novel function of CD34 in vitro. + The initial reconstitution model induces ASC expansion, and (3) the resulting induced CD34 + ASC(iCD34 + This provides the first evidence of a possible somatic tissue cell type derived from ASCs. + The in vitro expansion and derivation of ASCs provides unlimited and sufficient access to a patient's own adult stem cells in vitro and the ability to perform in vivo autologous transplantation therapies to treat a variety of human diseases, including, but not limited to, Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, AIDS, leukemia, lymphoma, many types of cancer, and aging. Autologous transplantation is the best method of stem cell therapy to prevent immune rejection. Consequently, this invention certainly lays a solid foundation for promoting the innovative development of novel stem cell therapies and will have a significant impact on the future of regenerative medicine.

[0015] Induced in vivo ASC expansion model Our first inducible in vivo ASC expansion model was established in 2011 using miR-302 precursor (pre-miR-302) isolated from iPSCs. Using a mouse skin wound healing model, we observed the development of ASC-like expanded pouches (still CD34 negative) in vivo after pre-miR-302 treatment (Chen et al., 2013; Lin SL, 2018). Because CD34 is a valid stem cell marker for human and porcine ASCs but not for mouse ASCs, we established a pig skin wound healing model in 2014 using the same treatment method and observed similar pouch-like CD34 negative cells in vivo. + As a result, there are now both porcine (CD34-positive) and murine (CD34-negative) ASC expansion models available to study the mechanisms of ASC expansion and induction in vivo, ex vivo, and in vitro.

[0016] Wound healing is the best in vivo model for studying the function of ASCs. Previous theories have suggested that ASCs can divide asymmetrically to generate one copy of themselves (ASC) and another differentiated daughter cell (called a progenitor cell), which can then further divide and differentiate into various new tissue cells that repair damaged tissue. Therefore, complete (intact) wound healing requires sufficient ASCs to provide abundant new tissue cells to completely repair and restore the entire wounded tissue area. Maintaining homeostasis of a sufficient ASC population in vivo is a critical step. However, the previous theory of "asymmetric stem cell division" fails to maintain such ASC homeostasis due to many internal and environmental risks that can damage ASCs, including but not limited to pollution, toxins, radiation, stress, injury, disease, and even aging. To overcome this problem, we attempted to induce and / or enhance symmetric ASC division using the DNA demethylation function of miR-302.

[0017] Using an in vivo pig skin wound healing model (Figures 1 and 3), we found that treatment with both isolated pre-miR-302 (miR-302) and iPSC lysate induced rapid and intact wound healing in vivo in all tested samples (n = 12 / 12 for each group), compared with the untreated control and other control treatments with miR-434-mock siRNA (miR-434). More specifically, Figure 5 further shows the histological results of intact wound healing after miR-302 treatment (i.e., sample 190-BR2) compared with the untreated control (i.e., sample 190-CR3). After all quantitative measurements, the line graphs in Figure 4 summarize the wound healing rates over time for the different treatment groups in Figure 3, respectively. Because the iPSC lysate also contains a large amount of miR-302, treatment with both miR-302 and iPSC lysate achieved a high wound healing rate of 90% on day 11, whereas other control treatments required an additional 6 days (day 17) to achieve the same healing results. Most importantly, during the first 3 days of wound healing, the miR-302 and iPSC lysate-treated specimens already showed over 10% to 20% wound contraction, which is the most critical step required for intact wound healing. Because wound contraction is known as a critical step in tissue repair during healing, delayed wound contraction can lead to disfigurement of the healed tissue and scar formation.

[0018] Using immunohistochemistry (IHC) staining with green fluorescent anti-CD34 antibody (Figure 5), we found expanded CD34 in skin treated with both miR-302 and iPSC lysates. + A regular distribution pattern of pouches of ASCs was observed, but not in untreated or miR-434-treated samples. Each expanded pouch contained approximately 20 to 1200 CD34 cells. + Asymmetric stem cell division cannot generate such a large number of ASC-expanded pouches, this must be due to the mechanism of "symmetric stem cell division" induced by miR-302 treatment. In particular, these CD34 +ASC pouches were regularly distributed across the dermal and epidermal junction, typically spaced approximately 240–290 μm apart, and expressed induced CD34 + This indicates that ASCs are specifically proliferating and expanding from the original ASCs in the skin tissue, and not random somatic cell reprogramming. + Although showing the same distance between ASCs, there is no sign of ASC expansion in the healed tissue area. This regular distance between the two ASCs may indicate an intact network of repair and restoration systems in the skin tissue.

[0019] From the results of Figures 1 to 5, our study demonstrated that (1) miR-302 treatment increased the wound healing rate by over 70 to 90% compared with conventional antibiotic ointment treatment (Figures 3 and 4), (2) miR-302 treatment led to very little or no wound healing in all tested specimens (n=12 / 12) (Figures 3 to 5), and (3) miR-302 treatment increased the CD34 expression in vivo in all treated specimens. + We confirmed that CD34 induces ASC expansion by more than 20-1000 fold (n=12 / 12) (Figure 5). Based on these findings, we now investigate the role of CD34 in vivo. + We concluded that the use of miR-302 under wound healing conditions is necessary to induce and maintain ASC expansion, resulting in perfect wound healing. It is believed that the combination of miR-302 with specific wound healing-related factors can induce and maintain ASC expansion in vitro. However, because there are many factors related to wound healing in vivo, the importance of each functional role in the mechanism of ASC expansion remains unclear. Therefore, the present invention identifies some of these specific factors.

[0020] To identify specific factors involved in ASC expansion, we further performed laser capture microdissection (LCM) and gene microarray analysis on isolated CD34 +We investigated the genetic profile of ASCs and the associated gene regulatory mechanisms of ASC expansion in vivo (Figures 1 and 2). Based on the identified genetic profile, we found that the process of induced ASC expansion and induction involves: (1) miR-302 downregulates several key epigenetic genes, particularly MECP1 / 2 and HDAC2 / 4, to establish a specific DNA demethylation pattern in the cellular genome; (2) the specific DNA demethylation pattern then induces the expression of a specific set of symmetric stem cell division (SSCD)-related genes and activates their downstream signaling pathways; and (3) the induced SSCD-related genes and specific wound healing-related factors cooperate to promote and maintain ASC expansion. However, most importantly, under specific DNA demethylation conditions, they continue to act cooperatively to complete the overall mechanism of ASC expansion and induction. From this newly established mechanism, all prior art methods clearly cannot provide such specific DNA demethylation conditions to sustain ASC expansion in vitro or ex vivo, and therefore cannot generate sufficient amplified ASCs required for clinical treatment. To overcome this problem, the present invention is the first method to use miR-302-mediated DNA demethylation to establish the conditions necessary to cooperate with specific wound healing-related factors to complete the entire process of ASC expansion and induction in vitro.

[0021] Induced in vitro CD34 + ASC expansion model Using LCM and microarray analysis (Figures 1, 2, and 6), we demonstrated that CD34 + Several specific factors that induce ASC expansion were identified. Initiating CD34 + ASCs can be isolated from enzymatically dissociated human or porcine skin cells (Figure 6, upper panel). Under feeder-free MSC expansion culture conditions supplemented with identified specific factors (e.g., bFGF / FGF2, LIF, and any other wound healing-related factors), treatment with miR-302 or its siRNA / shRNA mimics significantly increased the expression of isolated skin CD34 cells per in vitro treatment.+ We confirmed that ASC proliferation could be induced 20- to 100-fold (approximately ≥6-8 cell divisions in one cell culture passage). By refreshing the cell culture medium supplemented with fresh miR-302, LIF, bFGF / FGF2, and / or any other specific factors every 3-4 days, we were able to induce the proliferation of induced CD34 cells over 5-6 passages. + ASC(iCD34 + ASCs) can be repeatedly cultured, and ultimately the CD34 + Although LIF is one of the SSCD-related genes identified as necessary for ASC expansion, any other specific healing-related factor, including IGF, EGF, PDGF, VEGF, TGF, TNF, SCF, HOX, Notch, GSK, Wnt / β-catenin, interleukins, and / or BMPs, may be essential and enhance the effect of miR-302-induced ASC expansion.

[0022] Most interestingly, all expanded iCD34 + ASCs have a thin layer of CD34 + They are surrounded by an ASC-derived membrane (with weak or no CD34 expression) and are not in direct contact with other CD34-negative tissue cells (Figure 6, bottom left panel). As a result, pure iCD34 cells are available for further expansion. + ASC colonies can be easily isolated and collected. To date, iCD34 cells have been expanded for more than 10 passages without detectable changes in karyotype analysis. + The ASCs were successfully cultured and maintained. Subsequently, removal of miR-302 from the cell culture medium resulted in the iCD34 + The iCD34 induced ASC proliferation but did not affect cell viability (Figure 6, bottom right panel). +ASCs can further differentiate into several different tissue cell types, particularly in the ectodermal lineage in vitro (Figure 6, bottom right panel, including neural and skin cell types). For example, when both miR-302 and bFGF / FGF2 are removed, they differentiate into neural cells. Alternatively, when both miR-302 and LIF are removed, they differentiate into skin-type tissue cells. Isolated iCD34 + Further transplantation of ASCs into nude mice led to the formation of various tissue cell types in vivo, particularly in the ectodermal and mesodermal lineages (Figure 7). These findings suggest that the combination of miR-302 and certain factors can induce and maintain ASC expansion and induction in vitro, and that the induced CD34 expression may be useful for various cell therapies using expanded ASCs and / or ASC-differentiated tissue cells. + We conclude that this will lead to the establishment of a novel method for generating abundant and sufficient ASCs.

[0023] Previous studies of mouse and rat ESC regeneration have reported that the use of LIF / STAT3 and BMP4 / Id proteins can sustain ESC regeneration in vitro without the need for serum or feeders ( Ying et al., 2003 ; Xu et al., 2005 ), so CD34 + The recent discovery that LIF functions to induce ASC expansion indicates similar or parallel mechanisms shared by both ESC and ASC regeneration systems. Based on this situation, it is possible that LIF induces CD34 + While bFGF / FGF2 blocks the differentiation of ASCs into the mesodermal lineage, it also inhibits CD34 + We found that LIF and bFGF / FGF2 inhibited the differentiation of ASCs into the neuroectodermal lineage. +It blocks all differentiation potential of ASCs and maintains stem cell pluripotency. However, unlike ESCs, most ASCs do not strongly co-express Oct3 / 4 and Nanog, which would allow them to avoid replicative senescence. Therefore, the key question here is how ASCs avoid replicative senescence and achieve a long, unlimited life cycle. Previous studies (Lin et al., 2011; Lin and Ying, 2013) further demonstrated that miR-302 directly suppresses AOF2 / KDM1, DNMT1, and HDAC2 / 4, enhancing telomerase reverse transcriptase (TERT) activity and preventing iPSC senescence. Therefore, the SSCD genes identified by the microarray in this study, including but not limited to AOF2 / KDM1, DNMT1, HDAC2 / 4, and their downstream TERT, were identified as iCD34. + iCD34 protects against ASC senescence and inhibits ASC senescence in in vitro cell cultures + This suggests that it has the potential to extend the replicative life and passaging of ASCs.

[0024] iCD34 + In vivo transplantation model demonstrating ASC pluripotency induction The endogenous environment of different tissues / organs determines iCD34 in vivo + iCD34 is thought to guide and influence the cell fate of ASC differentiation. + To measure ASC differentiation, we utilized an in vivo transplantation model using NOD-SCID mice (n = 3). To track cell distribution and differentiation in vivo, we transfected these iCD34 cells with the red fluorescent protein (RFP)-expressing lentiviral vector pLVX-EF1a-HcRed-N1 (Clontech). + After labeling ASCs, RFP-positive CD34 + ASCs were selected by flow cytometry. Approximately 5 × 10 5 RFP-labeled iCD34 + ASCs were injected into the tail vein of NOD-SCID mice to complete the transplantation. Three weeks after transplantation, numerous RFP-positive CD34 +ASCs and their differentiated cells have been observed to survive and grow in many tissues and organs in vivo, including bone marrow, brain, heart, lung, spleen, and thyroid, and most are derived from either ectodermal or mesodermal lineages, with a few derived from endodermal lineages. As a result, Figure 7 shows that transplanted CD34 + We clearly demonstrate that ASCs and their differentiated tissue cells are abundant in bone marrow, brain, and thyroid tissues in vivo.

[0025] In particular, transplanted iCD34 + ASCs can survive and form numerous stem cell pouches / niches in the bone marrow, where hematopoietic stem cells (HSCs) also reside. However, these two types of CD34 + Whether and how ASCs are involved remains unclear. Differentiated RFP-positive blood cells were found in the peripheral bloodstream even 3 weeks after transplantation, as confirmed by blood smear examination under a fluorescent microscope, suggesting that transplanted iCD34 + These results suggest that ASCs can differentiate into at least some types of blood cells in vivo. HSCs and transplanted iCD34 cells are highly complementary in providing the body with various hematopoietic cell types in vivo. + If the physiological function of ASCs is replaceable, the patient's own iCD34 + It is conceivable that ASCs could be used to treat many blood-related diseases, including but not limited to stroke, myocardial infarction, AIDS, leukemia, lymphoma, and sickle cell anemia, among others, and such autologous transplantation is the best method of treatment to prevent immune rejection.

[0026] Furthermore, as shown in Figure 7, iCD34 in the brain + The discovery of RFP-positive neurons associated with the ASC pouch is another important rationale for developing novel therapeutic approaches to treat various degenerative nerve-related diseases, including but not limited to stroke, diabetes, dementia, Alzheimer's disease, Parkinson's disease, and motor neuron disease.

[0027] Current knowledge about ASC lifespan and its unique method of regulating cell division (i.e., asymmetric vs. symmetric stem cell division) is very limited. + It is unclear whether there are differences and / or similarities between ASC types. To resolve these issues, the current in vitro and in vivo CD34 + The ASC expansion model will be a useful tool to investigate these issues. + Using an ASC expansion model, (1) miR-302 inhibited CD34 expression in vivo. + It has the ability to induce ASC expansion and plays an important role in complete wound healing. (2) The use of miR-302 and certain factors, such as bFGF / FGF2 and LIF, has been shown to enhance the proliferation of CD34 in vitro. + It can induce ASC expansion and has many research and medical uses for amplified iCD34 + (3) providing ASCs and expanding iCD34 + In vivo transplantation of ASCs can grow and form various differentiated tissue cells in many organs / tissues, and these iCD34 + ASCs exhibit pluripotency, particularly in the ectodermal and mesodermal lineages, and (4) transplanted iCD34 + ASCs have been found to grow and form pouch-like stem cell niches in the bone marrow, brain, spleen, and thyroid gland, and show great potential for developing novel regenerative medicines and therapies to help treat aging, as well as Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, AIDS, leukemia, lymphoma, and many types of cancer.

[0028] A.Definition To facilitate the understanding of this invention, a number of terms are defined below. nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogen-containing heterocyclic base. A base is linked to the sugar moiety via the glycosidic carbon (the 1' carbon of the pentose), and the combination of base and sugar is a nucleoside. A nucleoside containing at least one phosphate group attached to the 3' or 5' position of the pentose is a nucleotide. DNA and RNA are composed of different types of nucleotide units called deoxyribonucleotides and ribonucleotides, respectively.

[0029] Oligonucleotides Oligonucleotides: Molecules containing two or more, preferably three or more, and usually ten or more, DNA and / or RNA. Oligonucleotides longer than 13 nucleotide monomers are also called polynucleotides. Their exact size depends on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced by any method, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.

[0030] Nucleotide Analogues : A purine or pyrimidine nucleotide that differs in structure by adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U) but is sufficiently similar to replace the normal nucleotide in a nucleic acid molecule.

[0031] Nucleic acid composition Nucleic acid compositions refer to oligonucleotides or polynucleotides, such as DNA or RNA sequences in single- or double-stranded molecular configurations, or mixed DNA / RNA sequences.

[0032] gene: A nucleic acid composition in which an oligonucleotide or polynucleotide sequence encodes RNA and / or polypeptide (protein). A gene may be RNA or DNA. A gene may encode non-coding RNA such as small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors and derivatives. Alternatively, a gene may encode protein-coding RNA important for protein / peptide synthesis, such as messenger RNA (mRNA) and its RNA precursors and derivatives. A gene may also encode protein-coding RNA that includes at least a microRNA or shRNA sequence.

[0033] Primary RNA transcript : an RNA sequence that is directly transcribed from a gene without RNA processing or modification, and may be selected from the group consisting of mRNA, hnRNA, rRNA, tRNA, snoRNA, snRNA, pre-microRNA, viral RNA and their RNA precursors and derivatives.

[0034] precursor messenger RNA (pre-mRNA) : The primary RNA transcription product of a protein-coding gene, generated in eukaryotes by the eukaryotic type II RNA polymerase (Pol-II) mechanism through the intracellular mechanism of transcription. The pre-mRNA sequence contains the 5'-untranslated region (UTR), 3'-UTR, exons, and introns.

[0035] Introns : A part of a gene's transcribed sequence that encodes a non-protein reading frame, such as an in-frame intron, 5'-UTR, or 3'-UTR. Exon : A portion of a gene transcribed sequence that encodes a protein reading frame (cDNA), for example, cDNA of cellular genes, growth factors, insulin, antibodies and their analogs / homologues and derivatives.

[0036] messenger RNA (mRNA): A collection of pre-mRNA exons, formed by the intracellular RNA splicing mechanism (spliceosome) after intron removal, which serves as protein-coding RNA for peptide / protein synthesis. Peptides / proteins encoded by mRNA include, but are not limited to, enzymes, growth factors, insulin, antibodies, and their analogs / homologues and derivatives.

[0037] complementary DNA (cDNA) : It is a single- or double-stranded DNA that contains a sequence complementary to the mRNA sequence and does not contain intron sequences. Sense : A nucleic acid molecule that has the same sequence order and composition as a homologous mRNA. The sense structure is indicated by the symbol "+", "s" or "sense".

[0038] antisense : A nucleic acid molecule complementary to the respective mRNA molecule. Antisense structures are indicated by a "-" or "*" symbol, or by "a" or "antisense" before the DNA or RNA, e.g., "aDNA" or "aRNA."

[0039] base pair (bp) Adenine (A) and thymine (T) or cytosine (C) and guanine (G) in double-stranded DNA molecules. In RNA, uracil (U) substitutes for thymine. This combination generally occurs through hydrogen bonds.

[0040] base pair (bp) Adenine (A) and thymine (T) or cytosine (C) and guanine (G) are paired in double-stranded DNA molecules. In RNA, uracil (U) substitutes for thymine. This pairing is generally achieved through hydrogen bonding. For example, the sense base sequence 5'-A-T-C-G-U-3' can form a perfect base pair with its antisense sequence 5'-A-C-G-A-T-3'.

[0041] 5' endA nucleotide-free end of consecutive nucleotides in which the 5'-hydroxyl group of one nucleotide is joined by a phosphodiester linkage to the 3'-hydroxyl group of the next nucleotide. One or more other groups, such as phosphates, may be present at the end.

[0042] 3' end A nucleotide-free end at the 3' position of consecutive nucleotides in which the 5'-hydroxyl group of one nucleotide is joined by a phosphodiester linkage to the 3'-hydroxyl group of the next nucleotide. Another group, usually a hydroxyl group, may be present at the end.

[0043] template A nucleic acid molecule being replicated by a nucleic acid polymerase. The template can be single-stranded, double-stranded, or partially double-stranded, depending on the polymerase. The synthesized copy will be complementary to the template or at least one strand of a double-stranded or partially double-stranded template. Both RNA and DNA are synthesized in the 5' to 3' direction. The two strands of a double-stranded nucleic acid are always aligned so that the 5' ends (and, optionally, the 3' ends) of the two strands are at opposite ends of the duplex.

[0044] be saved : A base sequence is conserved relative to a preselected (reference) sequence when it hybridizes non-randomly to the exact complement of that preselected sequence. Homolog or Homology : A term indicating the similarity between a polynucleotide and a gene or mRNA sequence. A nucleic acid sequence may be partially or completely homologous (homologous) to, for example, a gene or mRNA sequence. Homology may be expressed as a percentage determined by the number of similar nucleotides relative to the total number of nucleotides.

[0045] complementary or complementarity or complementarity: A term referring to the matching base pairs between two polynucleotides (i.e., mRNA and cDNA sequences) referred to by the "base pair (bp)" rules above. For example, the sequence "5'-A-G-T-3'" is complementary to the sequence "5'-A-C-T-3'" and also to "5'-A-C-U-3'." Complementarity can exist between two DNA strands, a DNA strand and an RNA strand, or two RNA strands. Complementarity can be "partial," "complete," or "full." Partial complementarity or complementarity occurs when only some of the nucleic acid bases match according to the base-pairing rules. Full complementarity or complete complementarity occurs when the bases between nucleic acid strands match completely or perfectly. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important for amplification reactions and detection methods that rely on binding between nucleic acids. Percent complementarity, or complementarity, is the number of mismatched bases relative to the total number of bases in a strand of nucleic acid. Thus, 50% complementarity means that half the bases are mismatched and the other half are matched. Two strands of nucleic acid can be complementary even if they have a different number of bases. In this case, complementarity occurs in the portion of the longer strand that corresponds to a base in the strand that pairs with a base in the shorter strand.

[0046] complementary bases :A nucleotide that usually pairs when DNA or RNA adopts a double-stranded structure. Complementary nucleotide sequence : A sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to a sequence of nucleotides in another single strand to specifically hybridize the two strands with the resulting hydrogen bonds.

[0047] Hybridization and hybridizationCompetitive inhibition: The formation of a duplex between nucleotide sequences that are sufficiently complementary to form a complex through base pairing. A primer (or splice template) "hybridizes" with a target (template), and such a complex (or hybrid) becomes sufficiently stable to perform the priming function required by DNA polymerase to initiate DNA synthesis. There is a specific, i.e., non-random, interaction between two complementary polynucleotides that is competitively inhibited.

[0048] Post-transcriptional gene silencing : A knockout or knockdown effect of a target gene at the level of mRNA degradation or translational repression, usually caused by either a foreign / viral DNA or RNA transgene or a small inhibitory RNA molecule.

[0049] RNA interference (RNAi) : A mechanism of post-transcriptional gene silencing in eukaryotes, mediated by small inhibitory RNA molecules, such as microRNAs (miRNAs), small hairpin RNAs (shRNAs), and small interfering RNAs (siRNAs). These small RNA molecules typically function as gene silencers, interfering with the expression of cellular genes that are fully or partially complementary to the small RNA.

[0050] Gene silencing effect : A cellular response after gene function is suppressed, consisting of, but not limited to, cell cycle attenuation, G0 / G1-checkpoint arrest, tumor suppression, anti-tumorigenicity, cancer cell apoptosis, and combinations thereof.

[0051] Non-coding RNA (ncRNA):Non-coding RNAs are RNA transcripts that are not used to synthesize peptides or proteins via the intracellular translation mechanism. Non-coding RNAs include long and short regulatory RNA molecules, such as microRNAs (miRNAs), small hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), and double-stranded RNAs (dsRNAs). These regulatory RNA molecules typically function as gene silencers, interfering with the expression of cellular genes that are fully or partially complementary to the non-coding RNA.

[0052] MicroRNA (miRNA) MicroRNAs are single-stranded RNAs that can bind to target gene transcripts (mRNAs) that share partial sequence complementarity with the microRNA. Mature microRNAs are typically approximately 17–27 oligonucleotides in length and, depending on the complementarity between the microRNA and its target mRNA, can either directly degrade intracellular mRNA targets or inhibit the translation of their target mRNA into protein. Natural microRNAs are found in nearly all eukaryotic organisms, function as defenses against viral infections, and enable the regulation of specific gene expression in plant and animal development. While a single microRNA often targets multiple target mRNAs to achieve full functionality, multiple miRNAs can also target the same gene transcript to enhance gene silencing effects.

[0053] Pre-miRNA (Pre-miRNA) Pre-miRNAs are hairpin-shaped single-stranded RNAs with stem arms and stem-loop regions that interact with the intracellular RNase III Dicer endoribonuclease to generate one or more mature microRNAs (miRNAs) that can silence target genes or specific groups of target genes that are fully or partially complementary to the mature microRNA sequence. The stem arms of pre-miRNAs can form perfect (100%) or partial (mismatched) double-stranded hybrids, while the stem-loop connects one end of the double-stranded stem arms to form a circular or hairpin-loop structure required for incorporation into the RNA-induced silencing complex (RISC) by Argonaute proteins (AGO).

[0054] Small interfering RNA (siRNA) : A short double-stranded RNA approximately 18-27 perfectly base-paired double-stranded ribonucleotides in size that can degrade target gene transcripts with near-perfect complementarity.

[0055] Small molecules or short hairpin RNAs (shRNAs) miRNAs are single-stranded RNAs containing a pair of partially or perfectly matched stem-arm nucleotide sequences that are split by a mismatched loop oligonucleotide to form a hairpin-like structure. Many natural miRNAs are derived from hairpin-shaped RNA precursors, or precursor microRNAs (pre-miRNAs).

[0056] vectorA vector is a recombinant nucleic acid composition, such as recombinant DNA (rDNA), capable of movement and residence in different genetic environments. Generally, other nucleic acids are operatively linked to it. A vector is capable of autonomous replication in cells in which the vector and its attached segments are replicated. One type of preferred vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication. Preferred vectors are those capable of autonomous replication and expression of nucleic acids. Herein, a vector capable of directing the expression of a gene encoding one or more polypeptides and / or non-coding RNA is referred to as an "expression vector" or "expressible vector." A particularly important vector allows for the cloning of cDNA from mRNA generated by reverse transcriptase. The vector may contain components consisting of a viral or type II RNA polymerase (Pol-II or Pol-2) promoter, or both, a Kozak consensus translation initiation site, a polyadenylation signal, multiple restriction / cloning sites, a pUC origin of replication, an SV40 early promoter for expressing at least a drug resistance gene in replicating prokaryotic cells, an optional SV40 origin for replication in mammalian cells, and / or a tetracycline responsive element. The vector structure may be linear or circular, single- or double-stranded DNA selected from the group consisting of plasmids, viral vectors, transposons, retrotransposons, DNA transgenes, jumping genes, and combinations thereof.

[0057] promoter A promoter is a nucleic acid that a polymerase molecule recognizes or binds to and initiates RNA transcription. For purposes of this invention, a promoter may be any sequence that can initiate synthesis of an RNA transcript with a desired polymerase, such as a known polymerase or its cofactor binding site, a promoter, etc.

[0058] Cisteron : A nucleotide sequence in a DNA molecule that encodes an amino acid residue sequence and includes upstream and downstream DNA expression control elements. Intron Excision: The cellular mechanisms responsible for RNA processing, maturation, and degradation, including RNA splicing, exosome digestion, nonsense-mediated decay (NMD) processing, and combinations thereof.

[0059] RNA processing : The cellular mechanisms responsible for RNA maturation, modification, and degradation, including RNA splicing, intron excision, exosome digestion, nonsense-mediated decay (NMD), RNA editing, RNA processing, and combinations thereof.

[0060] target cell The cells are one or more human cells selected from the group consisting of somatic cells, tissues, stem cells, germ cells, teratoma cells, tumor cells, cancer cells, and combinations thereof. cancer tissue The tumor tissue is derived from the group consisting of: skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor / carcinoma, lymphoma, leukemia, and combinations thereof.

[0061] gene delivery The method of genetic engineering is selected from the group consisting of: polysome transfection, liposome transfection, chemical transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, biolistic entry, and combinations thereof.

[0062] genetic engineering The DNA recombination method is selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene integration, transposon insertion, gene jump integration, retroviral infection, and combinations thereof.

[0063] Cell Cycle Regulators : Cellular genes involved in controlling cell division and proliferation rate, including, but not limited to, CDK2, CDK4, CDK6, cyclin, BMI-1, p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1, p27Kip1, and combinations thereof.

[0064] Tumor suppression effect Cellular anti-tumor and / or anti-cancer mechanisms and responses, including, but not limited to, cell cycle attenuation, cell cycle arrest, tumor cell growth inhibition, inhibition of cellular tumorigenicity, inhibition of tumor / cancer cell transformation, induction of tumor / cancer cell apoptosis, induction of normal cell restoration, reprogramming of high-grade cancer cells to a more benign, low-grade state (tumor regression), and combinations thereof.

[0065] Cancer treatment effects : A cellular response and / or cellular mechanism resulting from drug treatment, including, but not limited to, suppression of oncogene expression, suppression of cancer cell proliferation, suppression of cancer cell invasion and / or migration, suppression of cancer metastasis, induction of cancer cell death, prevention of tumor / cancer development, prevention of cancer recurrence, suppression of cancer progression, repair of damaged tissue cells, reprogramming of high-grade cancer to a more benign, low-grade state (cancer regression / remission), and combinations thereof.

[0066] Gene silencing effect : A cellular response following the suppression of gene function, consisting of, but not limited to, the suppression of oncogene expression, the suppression of cell proliferation, cell cycle arrest, tumor suppression, cancer regression, cancer prevention, cell apoptosis, cell repair and / or activation, cell reprogramming, reprogramming of diseased cells to a relative normal state (natural healing), and combinations thereof.

[0067] Cancer Reversal : A reprogramming mechanism that resets the malignant properties of aggressive cancers to a relatively normal, low-grade state in vitro, ex vivo, or in vivo. antibody : A peptide or protein molecule with a preselected, conserved domain structure that encodes a receptor capable of binding a preselected ligand.

[0068] Human Degenerative Diseases (HDD): HDD includes, but is not limited to, Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, leukemia, lymphoma, many types of cancer and age-related diseases.

[0069] Pharmaceutical and / or therapeutic applications Biomedical applications, devices and / or apparatus useful in: diagnostics, stem cell generation, stem cell research and / or therapeutic development, tissue / organ repair and / or rejuvenation, wound healing treatment, tumor suppression, cancer treatment and / or prevention, disease therapy, drug production, and combinations thereof.

[0070] B. Composition and Applications The present invention provides a composition and method for inducing the expansion and / or induction of adult stem cells (ASCs) in vitro using miR-302-like pre-miRNA, shRNA, and / or siRNA having the shared sequence 5'-UAAGUGCUUC CAUGUUU-3' (SEQ. ID. NO. 7) at its 5' end, in combination with specific factors related to wound healing, including, but not limited to, basic fibroblast growth factor (bFGF) / fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), stem cell factor (SCF), homeobox protein (HOX), Notch, GSK, Wnt / beta-catenin signaling, interleukins, and / or bone morphogenetic proteins (BMPs). The resulting expanded ASCs are useful for treating a variety of aging and cell damage-related human diseases, including but not limited to Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, AIDS, leukemia, lymphoma, many types of cancer, and aging.

[0071] In principle, the present invention relates to a novel mechanism of inducible symmetric ASC division recently discovered in an in vivo skin wound healing model (Lin, US Pat. No. 9,879,263 and US Patent Application No. 15 / 661,346). However, our previous invention does not reveal the specific factors that must be used together with miR-302 to induce ASC expansion in vitro. To overcome this problem, the present invention further discloses all essential and optional specific factors involved in the mechanism of induced ASC expansion in vitro. [Brief explanation of the drawings]

[0072] This patent or application contains at least one color drawing. Copies of this patent or patent publication with color drawing(s) will be provided by the USPTO upon request and payment of the necessary fee.

[0073] In particular, by way of example only and not by way of limitation, the drawings are as follows: [Figure 1] Figure 1 shows a proposed model of the miR-302-mediated intact wound healing mechanism in vivo. [Figure 2] Figure 2 illustrates the LCM-microarray analysis technique for identifying differentially expressed genes in two different tissue samples isolated in vivo. For example, a laser capture microdissection instrument (LCM) can be used to collect two different types of tissue cells from different sample regions. Then, mRNA / cDNA libraries are separately amplified and collected from each LCM-dissected tissue cell sample, and then further analyzed using microarrays. Using this LCM-microarray approach, we study and compare the differential gene expression patterns between in vivo-isolated CD34+ ASCs, in vitro-induced iCD34+ ASCs, and other non-ASC tissue cells. [Figure 3]Figure 3 shows the in vivo wound healing rate comparison between treatment with antibiotic ointment alone (top row), antibiotic ointment and 1 mg / mL miR-434-mock siRNA (second row), antibiotic ointment and 1 mg / mL isolated miR-302 precursor (third row), and antibiotic ointment and 5 mg / mL isolated iPSC lysate (bottom row). Sample numbers were n = 12 for the miR-302 and iPSC lysate treatment groups, and n = 6 for the blank control (ointment only) and miR-434 treatment groups. [Figure 4] Figure 4 shows the time course of wound healing rates (%) after treatment with (A) iPSC lysate (blue diamonds), (B) miR-302 precursor (miR-302; red squares), (C1) antibiotic ointment only (blank control; green triangles), and (C2) miR-434-mock siRNA (purple crosses). The results demonstrate that both iPSC lysate and miR-302 treatment significantly improved the rate of rapid wound healing in vivo (p<0.01), but not the other controls. [Figure 5]Figure 5 shows the contrast of wound healing outcomes between untreated (top, 190-CR3) and miR-302-treated (bottom, 190-BR2) in vivo skin samples. Isolated pre-miR-302 (20–1000 μg / mL) was formulated with antibiotic ointment and applied directly to a 2 cm x 2 cm open wound in the skin of a pig's back in vivo. Approximately 2–3 weeks after treatment, some of the healed wound samples were minced and further processed for histological examination. The results show that the miR-302-treated samples (perfect healing rate, n = 6 / 6) had no or very little scarring, while most untreated wounds (treated with antibiotic ointment only) had extensive scarring. Notably, significantly higher (≥40-1000-fold higher) CD34-positive adult stem cell populations (CD34+ASCs labeled with green fluorescent antibodies) were found in miR-302-treated specimens (n=6 / 6) compared with untreated control wounds. These results suggest that miR-302 can enhance tissue repair and regeneration by inducing and expanding CD34+ASCs, potentially offering a particularly advantageous therapeutic effect for lesions resulting from human degenerative diseases (HDD). [Figure 6] Figure 6 shows the results of induced in vitro CD34+ ASC expansion and induction. After skin cell dissociation and further serial dilution to obtain single cell colonies, CD34+ ASCs were identified using anti-CD34 antibody fluorescent immunocytochemical staining (top panel; green). These isolated CD34+ ASCs were then repeatedly cultured and expanded in vitro under established culture conditions containing miR-302 and identified specific factors (e.g., bFGF / FGF2, LIF, and any other wound healing-related factors) (bottom left panel). The expanded CD34+ ASCs (referred to as iCD34+ ASCs) could be further differentiated into several different tissue cell types in the skin and neuroectodermal lineages (bottom right panel) (n = 25, p < 0.001). [Figure 7]Figure 7 shows the results of in vivo transplantation of iCD34+ ASCs into NOD-SCID mice (n=3). 5×105 iCD34+ ASCs were transplanted into each mouse via tail vein injection. All iCD34+ ASCs were labeled with red fluorescent protein (RFP) delivered by the pLVX-EF1alpha-HcRed-N1 lentiviral vector. Approximately 3 weeks after transplantation, all major organs and tissues were collected separately and prepared into histological slides. IHC staining and microscopy were used to confirm the presence of the transplanted iCD34+ ASC-expanded pouch and iCD34+ ASC-differentiated tissue cells (all labeled red) in vivo. While three major tissue types are shown here, the types of tissue cells differentiated by iCD34+ ASCs observed in the current results include, but are not limited to, bone marrow, brain, heart, lung, spleen, thyroid, kidney, and liver. Among these, most are derived from the ectoderm or mesoderm, with fewer from the endodermal lineage. DETAILED DESCRIPTION OF THE INVENTION

[0074] In the experimental disclosure that follows, the following abbreviations apply: M (mole), mM (millimolar), μm (micromole), mol (mole), pmol (picomole), gm (gram), mg (milligram), μg (microgram), ng (nanogram), L (liter), ml (milliliter), μl (microliter), °C (degrees Celsius), RNA (ribonucleic acid), DNA (deoxyribonucleic acid), dNTP (deoxyribonucleotide triphosphate), PBS (phosphate buffer), NaCl (sodium chloride), HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), HBS (HEPES buffer), SDS (sodium dodecyl sulfate), TrisHCl (trishydroxymethylaminomethane hydrochloride), ATCC (American Type Culture Collection, Rockville, MD), hESC (human embryonic stem cells), and iPSC (induced pluripotent stem cells).

[0075] 1. In Vitro CD34 + ASC isolation, culture and expansion Starting CD34 + ASCs can be obtained from hair follicles according to our protocol (Lin et al., 2011), from enzymatically dissociated skin cells using Aasen's protocol (Nat. Protocols 5, 371–382, 2010), or simply from heparin-treated buffy coat tissues surrounding blood cells. Tissue samples should be kept fresh and immediately treated with a mixture of 4 mg / mL collagenase I and 0.25% TrypLE for 15–45 min depending on cell density. After washing twice with HBSS containing trypsin inhibitor, the samples should be transferred to a new sterile microtube containing 0.3 mL of feeder-free MSC expansion SFM culture medium (ASC culture medium, Irvine Scientific, CA). The cells were then further dissociated by shaking in a microtube incubator at 37°C for 1 min, and 0.3 mL of the entire cell suspension was transferred to a 35 mm Matrigel-coated culture dish containing 1 mL of feeder-free MSC expansion SFM culture medium supplemented with formulated miR-302 / pre-miR-302, LIF, and bFGF / FGF2, or any other specified factors. The concentrations of miR-302 / pre-miR-302, LIF, bFGF / FGF2, and any other specified factors used are 0.001 micrograms / mL to 500 micrograms / mL (0.001-500 μg / mL), respectively. Most preferably, the concentrations used in the ASC culture medium are 10-200 micrograms / mL (10-200 μg / mL) for miR-302 / pre-miR-302 and 2-20 nanograms / mL (2-20 ng / mL) for LIF, bFGF / FGF2, and / or any other specified factors. The ASC culture medium and all supplements should be renewed every 3-4 days. CD34 +ASCs can be grown into numerous pouch-like expanded colonies, collected separately, and passaged to approximately 50%-60% confluence by exposing the cells to TrypLE for 1 minute and washing twice with HBSS containing trypsin inhibitor. For further ASC expansion, the isolated CD34 cells can be cultured in fresh ASC culture medium supplemented with formulated pre-miR-302, LIF, bFGF / FGF2, and / or any other specific factors. + ASCs were replated at dilutions ranging from 1:5 to 1:500.

[0076] 2. Isolation and Preparation of MicroRNAs and Pre-miRNAs Native miR-302 and pre-miR-302 can be extracted from the cytoplasm of either hESCs or iPSCs, or both, according to Lin SL's protocol (Lin SL, 2018). To collect the cytoplasm, ESCs or iPSCs are disrupted by ultracentrifugation at 17,500 g for 30 minutes at 4°C, and the suspension is then filtered through a 0.01 micron ultrafilter column (30 kDa / 100 nucleotide cutoff, Amicon Ultra-0.5 30K) according to the manufacturer's instructions (Millipore, Billerica, MA). Approximately 0.8–1 mL of ESC or iPSC cytoplasm can be recovered from 1–1.2 billion ESCs or iPSCs, respectively. To extract miR-302 and pre-miR-302, ESC or iPSC cytoplasm was further purified through a 0.001 micron nanofilter column (3 kDa / 10 nucleotide cutoff, Amicon Ultra-0.5 3K) and reconstituted from the flow-through. Meanwhile, all small RNAs were collected on the nanofilter and dissolved in DEPC-treated ddH2O (pH 5.5-5.6) by double autoclaving for further purification by high-performance liquid chromatography (HPLC). The size of the small RNAs thus obtained ranged from approximately 10 to 110 nucleotides (or 3 to 30 kDa), including pre-miRNA / miRNA and several tRNAs. Using miRNA microarray analysis, we confirmed that over 90% of the isolated small RNAs were pre-miR-302 and miR-302, which are the most abundant and stable small RNAs in ESCs and iPSCs. Alternatively, synthetic miR-302-mimetic siRNAs and / or shRNAs may be used instead of natural miRNAs / pre-miRNAs.Furthermore, bacterial cells may be used instead of ESCs and iPSCs for the production and extraction of miR-302 / pre-miR-302.

[0077] 3. miRNA Microarray and RT-qPCR Analysis The purity and quantity of isolated small RNAs were assessed by 2%–3% low-melting-point agarose gel electrophoresis and spectrophotometric analysis at UV 260 nm / 280 nm (Bio-Rad, Hercules, CA). Approximately 10 μg of small RNA isolated from each sample was then subjected to microarray analysis by LC Sciences (San Diego, CA). Each microarray chip was hybridized with a single sample labeled with Cy3 or Cy5 dye. Background subtraction, data normalization, and statistical calculations were performed according to the manufacturer's protocol. For dual-sample measurements, p-value calculations were performed to generate a list of transcripts differentially expressed by 3-fold or more (yellow-red signals). RT-qPCR was performed using a set of TaqMan primers for hsa-miR-302a and the associated Real-Time PCR Kit (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. Signals were detected using an ABI7300 Real-Time PCR System (Applied Biosystems, Life Technologies).

[0078] 4.Immunostaining measurement Tissue samples were embedded, sectioned, and immunostained as previously described (Lin et al., 2008, 2010). The primary antibody was a green fluorescent dye-labeled CD34 antibody (Santa Cruz & Sigma). Alternatively, fluorescent dye-labeled goat anti-rabbit or horse anti-mouse antibodies were used as secondary antibodies (Invitrogen-Molecular Probes, Carlsbad, CA). Positive results were examined and analyzed at 100x or 200x magnification using a Fluorescence 80i microscope with a quantitative system and Metamorph imaging program (Nikon).

[0079] 5. Bisulfite DNA Sequencing Genomic DNA was isolated from 2,000,000 cells using a DNA isolation kit (Roche). 1 μg of isolated DNA was further treated with bisulfite (CpGenome DNA Modification Kit, Chemicon, Temecula, CA) according to the manufacturer's instructions. Bisulfite treatment converted all unmethylated cytosines to uracil, while methylated cytosines remained as cytosines. For bisulfite DNA sequencing, the promoter region of the gene Oct4 was amplified with PCR primers 5′-GAGGCTGGAG CAGAAGGATT GCTTTGG-3′ (SEQ ID NO. 12) and 5′-CCCTCCTGAC CCATCACCTC CACCACC-3′ (SEQ ID NO. 13). For PCR, bisulfite-modified DNA (50 ng) was mixed with primers (100 pmol total) in 1x PCR buffer, heated to 94°C for 2 minutes, and then immediately chilled on ice. Next, 25 cycles of PCR were performed using a high-precision amplification PCR kit (Roche), with 94°C for 1 minute and 70°C for 3 minutes. PCR products of the correct size were further fractionated by 3% agarose gel electrophoresis, purified with a gel extraction filter (Qiagen), and used for DNA sequencing. Subsequently, a detailed profile of DNA methylation sites was generated by comparing the unchanged cytosines in the converted DNA sequence with those in the unconverted DNA sequence.

[0080] 6. Flow Cytometry Cells were trypsinized, granulated, and fixed for 1 hour at -20°C by resuspension in 1 ml of pre-chilled 70% methanol in PBS. The cells were granulated, washed once with 1 ml of PBS, granulated again, and resuspended in 1 ml of 1 mg / ml propidium iodide, 0.5 μg / ml RNAse in PBS for 30 minutes at 37°C. Approximately 15,000 cells were then analyzed on a BD FACSCalibur (San Jose, CA). Cell doublets were excluded by plotting pulse width against pulse area and gating on single cells. Collected data were analyzed using the software package Flowjo with the "Watson Pragmatic" algorithm.

[0081] 7. Skin Wound Healing Model and In Vivo CD34 + ASC expansion Landrace is a breed of white, lop-eared pig most commonly found in Central and Eastern European countries. Male Landrace pigs used to establish the skin wound model were provided and cared for by the Animal Welfare Institute (ATIT), which is supervised by specially designated personnel and fully qualified veterinarians. Under their supervision, these animals were provided with appropriate care in accordance with Taiwan's Animal Welfare Act. These pigs were an average of 3 months old and weighed 18–23 kg. All animals were euthanized at the end of the study.

[0082] The animals were euthanized using Zoletil 50 (6 mg / kg) and their backs were shaved afterwards. Six (6) full-thickness square wounds (each 2 cm x 2 cm or 4 cm) were made with a sterile scalpel. 2Three wounds were created on the right and left sides of each animal. Each wound was treated topically with 0.5 mL of antibiotic ointment containing (A) iPSC lysate (5 mg / mL), (B) formulated miR-302 precursor (1 mg / mL), and (C1) blank or (C2) miR-434 siRNA (1 mg / mL). Treatments were performed on days 0, 1, 2, 3, 4, 5, 7, 9, 11, 14, and 17. Photographs of each wound were taken with a Sony DSC-H9 camera on days 0, 1, 2, 3, 4, 5, 7, 9, 11, 14, 17, and 20. The wound area at each time point was determined using the image processing software Image Pro Plus 7.0. The percentage of wound healing or union at each treatment time point was calculated according to the formula: (wound area on day 0 - wound area on day N) / wound area on day 0 × 100. Tissue samples were also collected from each wound and immersed in 10% (v / v) formalin solution before being used to prepare histological sections for H&E staining.

[0083] 8.Statistical analysis All data were expressed as the mean and standard deviation (SD). The mean value of each test group was calculated using AVERAGE in Microsoft Excel. SD was calculated using STDEV. Statistical analysis of the data was performed using one-way ANOVA. Tukey and Dunnett's t post hoc test was used to determine the significance of differences between the data groups. p<0.05 was considered significant (SPSS v12.0, Claritas Inc.).

[0084] References: 1. Aasen et al. (2010) Isolation and culture of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells, Nature Protocols 5, 371-382. 2. Chen SKJ and Lin SL, (2013) Recent patents on microRNA-guided pluripotent stem cell generation. Recent Patents on Regenerative Medicine 3:5-16. 3. Lin SL, Chang D, Chang‐Lin S, Lin CH, Wu DTS, Chen DT and Ying SY, (2008) Mir‐302 reprograms human skin cancer cells to a pluripotent ES cell‐like state, RNA 14, 2115–2124. 4. Lin SL and Ying SY, (2008) Role of the mir‐302 microRNA family in stem cell pluripotency and regeneration, in Ying SY, (Ed.) Current Perspectives in MicroRNAs, Springer-Verlag, New York, pp 167–185. 5. Lin SL, Chang D, Ying SY, Leu D and Wu DTS, (2010) MicroRNA miR-302 suppresses the tumorigenic potential of human pluripotent stem cells through coordinated suppression of the CDK2 and CDK4 / 6 cell cycle pathways. Cancer Res, 70, 9473-9482. 6. Lin SL, Chang D, Lin CH, Ying SY, Leu D and Wu DTS, (2011) Regulation of somatic cell reprogramming via inducible mir‐302 expression, Nucleic Acids Res, 39, 1054–1065. 7. Lin SL and Ying SY, (2013) Mechanism and method for generating tumor-free iPS cells by induction of intronic microRNA miR-302, Methods Mol Biol. 936, 295–312. 8. Lin SL, (2018) Identification and isolation of a novel sugar-like RNA-protecting material: glycylglycerol from pluripotent stem cells. Methods Mol Biol. 1733, 305-316. 9. Simonsson S and Gurdon J, (2004) DNA demethylation is required for epigenetic reprogramming of somatic cell nuclei, Nat Cell Biol. 6, 984–990. 10. Takahashi et al. (2006) Induction of pluripotent stem cells by specific factors from mouse embryonic and adult fibroblast cultures. Cell, 126, 663-676. 11. Wang et al. (2008) Embryonic stem cell-specific microRNAs regulate the G1-to-S transition and promote rapid proliferation. Nat. Genet. 40, 1478-1483. 12. Wernig et al. (2007) In vitro reprogramming of fibroblasts to a pluripotent ES cell-like state. Nature, 448, 318-324. 13. Xu RH, Peck RM, Li DS, Feng X, Ludwig T and Thomson JA (2005) Basic FGF and BMP signaling suppress undifferentiated proliferation of human embryonic stem cells. Nat Methods, 2, 185–190. 14. Ying QL, Nichols J, Chambers I and Smith A, (2003) BMP induction of Id proteins cooperates with STATs to suppress differentiation and continue self-renewal of embryonic stem cells, Cell, 115, 281-292. 15. Ying SY, Fang W and Lin SL, (2018) miR‐302‐mediated induced pluripotent stem cells (iPSCs): multiple synergistic reprogramming mechanisms, Methods Mol. Biol. 1733, 283–304. 16. Yu et al. (2007), Induced pluripotent stem cell lines derived from human somatic cells, Science, 318, 1917-1920. 17. Lin's European Patent EP2198025. 18. Lin, U.S. Patent No. 9,387,251. 19. Lin, U.S. Patent No. 9,394,538. 20. Lin, U.S. Patent No. 9,422,559. 21. Lin, U.S. Patent No. 9,567,591. 22. Lin, U.S. Patent No. 9,879,263. 23. Lin, U.S. Patent Application No. 12 / 318,806. 24. Lin, U.S. Patent Application No. 13 / 572,263.

Claims

1. CD34-positive adult stem cells (CD34 + 1. A method for inducing the expansion and induction of ASCs in vitro, comprising: (a) providing a small RNA comprising at least SEQ. ID. NO. 7; (b) providing specific factors associated with wound healing, including at least LIF and bFGF / FGF2; (c) at least isolated CD34-positive adult stem cells (CD34 + ASC), wherein the isolated CD34 + ASCs are adult stem cells isolated from human skin; (d) Under in vitro cell culture conditions, a small RNA comprising SEQ. ID. NO. 7 of (a), specific factors associated with wound healing including LIF and bFGF / FGF2 of (b), and isolated CD34 of (c). + ASCs were mixed with CD34 + Induction and maintenance of ASC population expansion and induction, and induced CD34 + and forming ASCs, wherein the induced CD34 + ASCs can form a stem cell niche.

2. The induced CD34 + The method of claim 1 , wherein the ASCs are capable of forming an adult stem cell niche or pouch in vivo after transplantation.

3. The induced CD34 + The method of claim 1 , wherein the ASCs can differentiate into multiple tissue cell types in the ectodermal or mesodermal lineage.

4. The induced CD34 + The method of claim 1, wherein the ASCs are multipotent both in vitro and after transplantation.

5. The method of claim 1, wherein the small RNA comprising SEQ. ID. NO. 7 comprises at least a hairpin stem-loop structure.

6. The method of claim 1, wherein the small RNA comprising SEQ. ID. NO. 7 comprises at least a hairpin-shaped microRNA precursor.

7. The method of claim 1, wherein the small RNA comprising SEQ. ID. NO. 7 is miR-302 precursor (pre-miR-302).

8. The method of claim 1, wherein the small RNA comprising SEQ.ID.NO.7 is SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, or SEQ.ID.NO.4, or a combination thereof.

9. The method of claim 1, wherein the small RNA comprising SEQ. ID. NO. 7 is siRNA.

10. The method of claim 1, wherein the small RNA comprising SEQ.ID.NO.7 comprises the sequence of SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10, or SEQ.ID.NO.

11.

11. The method of claim 1, wherein the small RNA comprising SEQ.ID.NO.7 can downregulate AOF2 (also known as KDM1 or LSD1), DNMT1, HDAC2 or HDAC4.

12. The method of claim 1, wherein the small RNA comprising SEQ. ID. NO. 7 is capable of causing DNA demethylation.

13. The small RNA comprising SEQ. ID. NO. 7 was isolated and induced + The method of claim 1, wherein the pluripotency of ASCs can be enhanced and maintained both in vitro and after transplantation.

14. The method of claim 1, wherein the specific wound healing-related factors including LIF and bFGF / FGF2 further include at least any factor selected from insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), stem cell factor (SCF), homeobox protein (HOX), Notch, GSK, Wnt / beta-catenin signaling, interleukin, and / or bone morphogenetic protein (BMP).

15. 2. The method of claim 1, wherein the in vitro cell culture conditions are feeder-free MSC expansion medium at 37°C.

16. The method of claim 1 , wherein the in vitro cell culture conditions further comprise Matrigel.

17. The induced CD34 + The method of claim 1 , wherein the ASCs aid in wound healing after transplantation.

18. The induced CD34 + 10. The method of claim 1, wherein the ASC is useful for the discovery of new drugs.

19. The induced CD34 + The method of claim 1, wherein the ASCs are useful for the development of pharmaceutical and / or therapeutic applications.

20. The induced CD34 + 10. The method of claim 1, wherein ASCs are useful for the development of therapeutics useful in the treatment of Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, osteoporosis, myocardial infarction, hemophilia, anemia, AIDS, leukemia, lymphoma, many types of cancer and aging.

21. CD34 + The method of claim 1, wherein ASC expansion is an inducible, symmetric adult stem cell division mechanism.

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