Significance In this report we describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor [platelet-derived growth factor–AB (PDGF-AB)] and 5-Azacytidine, a demethylating compound that is widely used in clinical practice. Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors. This method can be applied to both mouse and human somatic cells to generate multipotent stem cells and has the potential to transform current approaches in regenerative medicine.

Abstract Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

The goal of regenerative medicine is to reconstitute damaged or defective tissues. A supply of healthy cells capable of contributing to tissue repair and regeneration without risk of host rejection or malignant transformation is vital to achieving this goal. In this regard, bone marrow transplantation and skin and bone grafts have proven clinical utility (1⇓⇓–4). However, for the majority of tissues that require regeneration, harvesting and expanding stem and progenitor cells is a challenge, and tissue grafting is not an option due to site- and tissue-specific limitations. Although mesenchymal stem cells (MSCs) harvested from bone marrow or fat are regularly injected into sites of tissue injury, there is little objective evidence that these cells are retained at sites of injection or contribute directly to new tissue formation (5).

Given the challenges faced in harnessing resident stem cells to regenerate and repair tissues with low baseline turnover, research efforts over many decades have been directed toward converting terminally differentiated cells into stem cells. Somatic cells can be reprogrammed into pluripotent cells by transferring their nuclear contents into oocytes (6), by fusion with ES cells (7, 8), or by introducing defined transcription factors (9). However, as the direct implantation of autologous pluripotent cells runs the risk of ectopic tissue production and malignant transformation, the focus has shifted to reprogram cells directly into specific cell types using defined transcription factors (10). Methods to generate mouse [e.g., neurons (11), pancreatic b cells (12), hepatocyte-like cells (13), cardiomyocytes (14), and hematopoietic progenitors (15, 16)] and human [e.g., cardiomyocytes (17) and blood progenitors (4)] cells have been reported. However, these methods require vector-based gene transfer and are of low reprogramming efficiency. Moreover, most tissues are a complex mix of different cell types, and there is a need for the generation of autologous stem cells that can directly contribute to repair and regeneration of multiple cell types in a context-dependent manner.

Limb regeneration in salamander species is dependent not on resident stem-cell proliferation but plasticity of differentiated cells where mesenchymal tissues such as cartilage, muscle, and connective tissue underlying the wound epidermis lose their differentiated characteristics and adopt a blastemal cell state (18). The blastema is essentially a zone of mesenchymal cells that proliferate, differentiate, and regenerate the limb according to the predetermined body plan. To replicate the regenerative response in salamanders would require the generation of plastic, proliferative cells from terminally differentiated mammalian mesenchymal cell derivatives. During embryonic development, cells become more specialized, and their early transcriptional plasticity and multilineage developmental potential is gradually restricted by epigenetic silencing of genes (19). 5-Azacytidine (AZA) is a nucleoside analog that is used to treat blood disorders such as myelodysplasia and leukemia (20). AZA demethylates DNA (21) and as an inducer of cell plasticity (22) is used in protocols to transdifferentiate cells in vitro (23⇓⇓–26) and to convert partially reprogrammed induced-pluripotent stem (iPS) cells to fully reprogrammed iPS cells (27). However, the capacity of AZA to induce cell plasticity has not to our knowledge been harnessed to generate tissue-regenerative multipotent stem (iMS) cells.

We hypothesized that AZA-induced cell plasticity occurs via a transient multi- or pluripotent cell state and that concomitent exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues and report that AZA in combination with platelet-derived growth factor–AB (PDGF-AB) converts primary somatic cells into iMS cells. Importantly, unlike primary MSCs (5), which may facilitate but do not directly contribute to tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming teratomas.

Discussion iMS cells share a number of in vitro features with tissue-derived MSCs. However, iMS cells are transcriptionally distinct and have major in vivo advantages over MSCs, including their cellular plasticity, retention at graft sites, and contribution to tissue regeneration (SI Appendix, Table S1). The conversion process requires culturing cells or tissues transiently with AZA and PDGF-AB. AZA is administered to patients with myelodysplasia as a daily injection for 7 consecutive days (or interrupted by the weekend) followed by 3 wk off treatment (20). This 28-d cycle is repeated for as long as patients show benefit and often continues for many years. The dose of AZA (10 μM) that was required in conjunction with PDGF-AB to generate iMS cells was selected after testing concentrations within the peak plasma concentrations measured in patients (38, 39). AZA is incorporated into DNA and RNA (38), and PDGF-AB is both a mitogen and cell survival factor (40⇓–42). If PDGF-AB stimulation contributes to the uptake of AZA and proliferation of an otherwise transient cell population, it is unclear at this stage why PDGF-AB succeeds when the other cytokines do not. It is noteworthy that the endogenous expression patterns of PDGF-A and PDGF-B do not frequently overlap (40) and PDGF-AB heterodimers may be infrequent in vivo. It is possible that the early effect of AZA is to facilitate reexpression of genes that are required for cell conversion. For example, primary osteocytes lack PDGFRA expression, but as shown in Fig. 4E, PDGFRA-mediated signaling is required for cell conversion. Surface PDGFRA protein expression is induced by AZA and further enhanced by PDGF-AB, which requires PDGFRA for canonical signaling (SI Appendix, Fig. S20). Oct4 reexpression appears to be a key step in the evolution of somatic cells toward pluripotency (43). The exact sequence of events that leads to demethylation of the Oct4 promoter and Oct4 reexpression as well as nucleosome eviction at lineage-specific gene promoters following PDGF-AB/AZA treatment will require further investigation. Given the transcriptional connectivity of pluripotent genes (44), Oct4 reexpression in osteocytes by promoter demethylation may serve as the driver for reexpression of the others. The erasure of epigenetic barriers at lineage-committed genes is probably as important as the reactivation and low-level reexpression of pluripotent genes for the plasticity of these reprogrammed cells. The combination of PDGF-AB and AZA is also effective in converting primary human adipocytes in serum-free medium into proliferative CFU-Fs that can be extensively passaged in vitro (SI Appendix, Fig. S14). For both murine and human cells, further research is required to establish how transplanted iMS cells activate lineage-specific transcriptional programs in response to local cues and the range of tissues that can be generated in vivo. It will also be important to determine whether and for how long undifferentiated iMS cells remain dormant at sites of transplantation and retain their capacity to proliferate and differentiate on demand. Although there was no evidence of systemic spread or tumourigenicity at 12 wk, long-term evaluation will be necessary before clinical application. Nevertheless, this efficient vector-free method of generating autologous tissue-regenerative cells has significant merits over current approaches.

Materials and Methods Mice. All experiments involving mice were approved by the University of New South Wales animal ethics committee. The strains are detailed in SI Appendix. BmCFU-F Isolation and ex Vivo Expansion. BmCFU-Fs were isolated from wild-type C57BL/6, Q(S), Rag1, Pdgfrα-nGFP, or DMP1eYFP mice. Tibias and femurs were removed, cleaned of excess soft tissues, flushed out and thoroughly crushed using a mortar and pestle, and collagenase-treated, and the filtered supernatant was inactivated and plated in αMEM with 20% (vol/vol) FCS and penicillin/streptomycin/glutamine (P/S/G) (see SI Appendix). Primary Osteocyte Isolation and Culture. Osteocytes were isolated from long bones of 8–16-wk-old wild-type C57BL/6, Q(S), Rag1, or Pdgfrα-nGFP mice. Cells were FACS sorted for SCA1−/CD31−/PDGFRA−/CD51+ and cultured in DMEM + P/S/G + 100 μg/mL of ascorbate + 10% FCS (see SI Appendix). Primary Mature s.c. Adipocyte Isolation and Culture. Primary mature s.c. adipocytes were isolated from 8- to 16-wk-old PDGFRA-nGFP mice adipose tissue using a previously described method (45). Primary adipocytes were cultured in adipocyte medium (DMEM-HG + 10% FCS + P/S/G) at 37 °C and 5% CO 2 in the incubator for 8–10 d before exposing to reprogramming agents (see SI Appendix). Primary Osteocyte Isolation and Culture from DMP1-eYFP Mice. Primary osteocytes were isolated from long bones of 8–16-wk-old DMP1-eYFP mice. Cell suspensions from the primary isolation procedure and resulting bone fragments were cultured on type-I rat tail collagen-coated six-well plates in αMEM + 5% FCS + P/S/G for 7 d. Outgrowths of cells from bone fragments and cells in suspension were FACS-sorted for SCA1−/CD31−/PDGFRA−/DMP1eYFP+ and cultured in osteocyte culture media for 3 d before replacement with reprogramming medium (see SI Appendix). Cellular Reprogramming. Reprogramming in vitro-generated mouse osteocytes, chondrocytes, and adipocytes. In vitro-differentiated osteocytes, chondrocytes, and adipocytes were first cultured in MSC medium (αMEM + 20% FCS + P/S/G) with or without 10 μM AZA (Tocris Biosciences) and with or without cytokine (50 or 100 ng/mL Pdgf-AA or Pdgf-BB or Pdgf-AB, 10 ng/mL basic fibroblast growth factor, 20 ng/mL hepatocyte growth factor, 10 ng/mL insulin-like growth factor, and 10 ng/mL vascular endothelial growth factor; all from R&D Systems) for 2 d and then cultured in MSC media with or without cytokine for 10 d. To investigate the reprogramming cell-signaling pathways, inhibitors were added to the reprogramming mixture from day 1 and kept for 12 d. Media was refreshed every 3–4 d. At the end of day 12, cells were harvested for downstream analysis. Reprogramming primary mouse osteocytes. FACS-sorted long-bone–derived SCA1−/CD31−/PDGFRA−/DMP1eYFP+ primary osteocytes were cultured on type I rat tails in collagen-coated 35 mm2 dishes in the osteocyte culture medium (αMEM + 5% FCS + P/S/G) for 3 d. On day 4, osteocyte culture media was replaced with reprogramming media (αMEM + 20% FCS + 100 ng/mL mouse recombinant PDGF-AB + 10 μM AZA + P/S/G) and cultured for a further 48 h. At the end of 48 h, reprogramming media was replaced with αMEM + 20% FCS + 100 ng/mL mouse recombinant PDGF-AB + P/S/G media and cultured for a further 10 d with every 3 d media change. On day 12, reprogrammed cells were harvested for characterization. Reprogramming primary mouse adipocytes. Harvested primary adipocytes were cultured in adipocyte medium for 8–10 days. Reprogramming media [αMEM + 20% (vol/vol) FCS + 100 ng/mL mouse recombinant PDGF-AB + 10 μM AZA + P/S/G] was added on day 11 and cultured for further 48 h. At the end of 48 h, reprogramming media was replaced with αMEM + 20% FCS + 100 ng/mL mouse recombinant PDGF-AB + P/S/G media and cultured for another 10 d with every 3 d media change. On day 12, reprogrammed cells were harvested for characterization. Reprogramming primary human adipocytes. S.c. fat was harvested with consent from patients undergoing surgery for degenerative disk disease with approval from the Prince of Wales Hospital human research ethics committee. The s.c. adipocytes were harvested using a previously described method (45) with modifications (see SI Appendix). Harvested primary human adipocytes were exposed to reprogramming media containing 10 μM AZA + 200 ng/mL human recombinant PDGF-AB + 20% autologous serum + P/S/G for 2 d and then maintained in 200 ng/mL human recombinant PDGF-AB + 20% autologous serum + P/S/G for a further 23 d with every 3–4 d media change. On day 25, reprogrammed adipocytes were harvested for characterization. Inhibitor Assays. To investigate the reprogramming of cell signaling pathways, inhibitors were added to the reprogramming mixture from day 1 and kept for 12 d. Media was refreshed every 3–4 d. At the end of day 12, cells were harvested for downstream analysis (see SI Appendix). Live Cell Imaging. Cells were imaged using an IncuCyte microscope (Essen Bioscience) with 10× phase objective and a Nikon Ti-E microscope with a 20× phase objective (0.45 N.A.). Images were captured every 60 and 30 min, respectively, for 8 d (see SI Appendix). CFU-F Long-Term Growth and Serial Clonogenicity. For details on CFU-F long-term growth and serial clonogenicity, see SI Appendix. In Vitro Lineage Differentiation. Reprogrammed cells were differentiated into derivatives of all three germ layers as detailed in SI Appendix. Teratoma Formation. Rag1 mice were injected (under the kidney capsule) with mouse HM1 ES cells (n = 2) or CFU-Fs (n = 3), osteocytes (n = 3), and oCFU-Fs (n = 3) from β2-microglobulin-GFP mice either alone or as a mixture of mESCs and CFU-Fs (n = 3), osteocytes (n = 3), or oCFU-Fs (n = 3) (mESCs:cells, 1:3) (see SI Appendix). Immunohistochemistry. All antibodies and methods are listed in SI Appendix. Gene Expression and Epigenetic Analyses. All primers are listed under SI Appendix. High-quality RNA was profiled using Illumina’s Mouse WG-6 v2.0 Bead arrays and analyzed as detailed in SI Appendix. Allelic bisulphite sequencing and NOMe-Seq were performed as detailed in SI Appendix. Expression data have been deposited in the Gene Expression Omnibus under accession no. GSE59282. Posterior–Lateral Intertransverse Lumber Fusion Model. Long bones were harvested from either β2-microglobulin-GFP or DMP1eYFP mice; soft tissues were removed, flushed out, fragmented, and collagenase-treated; and the supernatant was discarded. Either bone fragments or DMP1eYFP+ osteocytes were cultured in reprogramming or control media for 12 d and surgically implanted into the posterior–lateral lumbar spine region (L4–L5) in Rag1 mice. At 6 and 12 wk, mice were euthanized and analyzed as detailed in SI Appendix.

Acknowledgments The authors thank Dr. C. Glenn Begley and Dr. Jose Polo for reading and commenting on the manuscript. This work was supported by grants from the National Health and Medical Research Council of Australia and the Australian Research Council. A.Y. was supported by an Endeavour Scholarship from the Australian Government.

Footnotes Author contributions: V.C., R.A.O., S.T.G., L.I., R.L.W., L.B.H., W.W., and J.E.P. designed research; V.C., A.Y., J.C.K., R.A.O., Q.Q., Y.C.K., P.Z., D.B., J.E.V., A.C.N., K.K., R.N., Y.Y., P.H., A.G., and F.D. performed research; R.M., C.R.W., and L.E.P. contributed new reagents/analytic tools; V.C., R.A.O., L.B., A.U., J.E.V., C.P., M.C., A.M., R.W., S.T.G., L.I., J.W.H.W., L.B.H., and J.E.P. analyzed data; and V.C. and J.E.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE59282).

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