Reprogramming of Skeletal Myoblasts for Induction of Pluripotency for Tumor-Free Cardiomyogenesis in the Infarcted HeartNovelty and Significance
Rationale: Skeletal myoblasts (SMs) with inherent myogenic properties are better candidates for reprogramming to pluripotency.
Objective: To reprogram SMs to pluripotency and show that reprogrammed SMs (SiPS) express embryonic gene and microRNA profiles and that transplantation of predifferentiated cardiac progenitors reduce tumor formation.
Methods and Results: The pMXs vector containing mouse cDNAs for Yamanaka's quartet of stemness factors were used for transduction of SMs purified from male Oct4-GFP+ transgenic mouse. Three weeks later, GFP+ colonies of SiPS were isolated and propagated in vitro. SiPS were positive for alkaline phosphatase, expressed SSEA1, and displayed a panel of embryonic stem (ES) cell–specific pluripotency markers. Embryoid body formation yielded beating cardiomyocyte-like cells, which expressed early and late cardiac-specific markers. SiPS also had an microRNA profile that was altered during their cardiomyogenic differentiation. Noticeable abrogation of let-7 family and significant up-regulation of miR-200a-c was observed in SiPS and SiPS-derived cardiomyocytes, respectively. In vivo studies in an experimental model of acute myocardial infarction showed extensive survival of SiPS and SiPS-derived cardiomyocytes in mouse heart after transplantation. Our results from 4-week studies in DMEM without cells (group 1), SMs (group-2), SiPS (group-3), and SiPS-derived cardiomyocytes (group 4) showed extensive myogenic integration of the transplanted cells in group 4 with attenuated infarct size and improved cardiac function without tumorgenesis.
Conclusions: Successful reprogramming was achieved in SMs with ES cell-like microRNA profile. Given the tumorgenic nature of SiPS, their predifferentiation into cardiomyocytes would be important for tumor-free cardiogenesis in the heart.
Cardiac regeneration using cell-based therapy is faced with the dilemma of identifying the ideal type of cells for transplantation into the infarcted heart. Besides suboptimal protocols of cell culture and transplantation, ease and availability of cells in sufficient number, their survival in the infarcted region and differentiation into morpho-functional cardiomyocytes for regeneration are some of the major challenges facing the heart stem cell therapy. Despite progress to clinical application, the use of stem cells has not been without controversies regarding their differentiation potential and ability to integrate with the host myocardium.1–4 Embryonic stem (ES) cells are the prototypical stem cells that unarguably possess near-ideal characteristics in terms of clonality, self-renewal, and multipotentiality.5 However, moral and ethical issues regarding availability and immunologic concerns have hampered their progress to clinical use.
The reprogramming of somatic cells with 4 stemness factors, called induced pluirpotent stem cells (iPS cells), has generated alternative sources of stem cells that possess characteristics reminiscent of ES cells in terms of cell biology and pluripotent differentiation characteristics, albeit with availability in large numbers without ethical issues and having better immunologic behavior.6 Although the main focus of the current research in iPS cell technology pertains to refinement of the protocols to enhance the efficiency of reprogramming and to circumvent the safety issues for their human use,7 there are few studies that have shown their regenerative potential in vivo in general and for the infarcted myocardium in particular. Terzic's group has taken the lead in this regard to show the reparability of mouse fibroblast-derived iPS cells in an immunocompetent mouse heart model of acute myocardial infarction.8 The authors have demonstrated that the transplanted undifferentiated iPS cells survived in the infarcted myocardium, and by 4 weeks, global heart function was recovered better than basal Dulbecco's modified Eagle's medium (DMEM)-injected control animals. The authors further reported no observation of teratomas in iPS cell–treated animal hearts. We have successfully generated mouse skeletal myoblast (SM)–derived iPS cells (SiPS), which expressed endogenous markers of stemness similar to mouse ES cells. We report that cardiomyocytes derived from the 10-day-old spontaneously beating embryoid bodies (EBs) obtained from SiPS (SiPS-CM) provide an excellent donor cell source that significantly attenuated infarct size expansion and improved contractile heart function in an experimental mouse model of acute myocardial infarction. Moreover, contrary to previous claims,8 we observed that SiPS transplanted in the infarcted heart of immunocompetent recipient were teratogenic.
Isolation of Mouse SMs
For our animal experiments, we used the Oct4/GFP transgenic mouse strain (Jackson Laboratories, Bar Harbor, ME) with GFP tagged to the endogenous Oct3/4 gene promoter. For SMs isolation, we followed the standard protocols routinely used in our laboratory as described in the Online Supplement at http://circres.ahajournals.org.9
Generation and Maintenance of SiPS
Retroviral vectors encoding for Yamanaka's quartet of pluripotentcy-determining factors were purchased from Addgene Inc. (Cambridge, MA) (Addgene plasmid #13367; #13366; #13370; #17220 from Takahashi et al).6 SMs from Oct4/GFP mice were seeded at a density of 1×105 cells/well in a 6-well plate. Twenty-four hours later, the cells were transduced with infectious supernatants from the respective vectors encoding for Oct4, Sox2, Klf4, and c-Myc factors for 48-hour transduction. Subsequently, the cells were replated in a 10-cm cell culture dish on mouse embryonic fibroblasts (MEFs) and observed for development of SiPS clones until 3 weeks. The GFP+ SiPS clones with ES cell-like morphology were mechanically incised, cultured on mouse feeder cells, and expanded individually in ES cell culture medium for use in further experiments. For confirmation of pluripotency induction, SiPS were fixed with 3% paraformaldehyde, permeabilized and stained with antistage-specific embryonic antigen-1 (SSEA-1) antibody. The primary antigen–antibody reaction was detected with goat antimouse Alexa Fluor-568 conjugated secondary antibody (1:200; Cell Signaling Tech, Danvers, MA). Nuclei were visualized by 4,6′-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA) staining. The murine SiPS clone Raf1 was expanded on mitotically inactivated murine embryonic fibroblasts (MEFs; 5×104 cells/cm2) as described in the Online Supplement.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Isolation of total RNA, and their subsequent first-strand cDNA synthesis, was performed using an RNeasy mini kit (Qiagen, Valencia, CA) and an Omniscript Reverse Transcription kit (Qiagen, Valencia, CA), respectively, per the instructions of the manufacturer and as described earlier.10 The primer sequences used are given in Online Table I.
Alkaline Phosphatase Staining and Immunocytochemistry
Alkaline phosphatase staining was done as per the manufacturer's instruction using Alkaline Phosphatase Detection kit (Millipore SCR004).
The undifferentiated colonies of iPS and ES cells were immunostained for the expression of the stage-specific embryonic antigen-1 (SSEA-1), Oct3/4, and Sox2 as described in the Online Supplement. The immunostained cells were observed and photographed with a microscope equipped for epifluorescence (Olympus, Tokyo, Japan).
Teratoma Formation and Karyotyping
Teratogenicity of SiPS was assessed in immunodeficient mice (n=3) as described in the Online Supplement. Karyotyping was carried out with quinacrine-Hoechst staining at the Transgenic Facility of the University of Kansas Medical Center (Kansas City, KS).
Spontaneous Cardiac Differentiation of SiPS
For differentiation into cardiomyocytes, SiPS were grown in suspension culture for 3 days in high-glucose DMEM containing 15% FBS, 5 mmol/L penicillin/streptomycin and 5 mmol/L nonessential amino acids. After 3 days in suspension culture, rounded EBs were formed, which were seeded on gelatin-coated dishes and cultured for 10 days. Adherent spontaneously contracting colonies were mechanically dissected and dissociated into single cardiomyocytes for use in further experiments.
miR Analysis of SiPS and SiPS-CM
Total RNA from SMs, SiPS, and SiPS-CM were labeled with Cy3. Samples were hybridized to a mouse miRNA microarray (LC Science, Houston, TX), according to manufacturer's protocol.
Ultrastructural studies were performed on the myocardial tissue samples as described in the Online Supplement using JEOL transmission electron microscope.
Experimental Animal Model and SiPS Engraftment
The present study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 to 23, revised 1985) and protocol approved by the Institutional Animal Care and Use Committee, University of Cincinnati.
A model of acute myocardial infarction was developed in allogenic 8- to 12-week-old female C57BL/6J immunocompetent mice as described earlier.11 Briefly, the animals were anesthetized (Ketamine/Xylazine 0.05 mL intraperitoneally), intubated, and mechanically ventilated (Harvard Rodent Ventilator, Model 683). Minimally invasive thoracotomy was performed for permanent ligation of coronary artery with a Prolene #9 to 0 suture. Myocardial ischemia was confirmed by color change of left ventricular wall. The animals were grouped (n=12 per group) for intramyocardial injection of 10 μL of basal DMEM without cells (group 1) or containing 3×105 SMs (group 2), SiPS (group 3), and SiPS-CMs (group 4). Additionally, young female transgenic Oct4/GFP (C57BL/6x129S4SV/jae) (n=4) animals were used for transplantation of SiPS in group 3 to ascertain whether the use of isogenic recipient could curtail tumorgenic potential of SiPS. The cells were injected 10 minutes after coronary artery ligation at multiple sites (3 to 4 sites per heart) in the free wall of the LV under direct vision. For postengraftment tracking of the transplanted cells and determination of their fate, the cells were labeled with Q-tracker®-625 (red fluorescence; Invitrogen, Carlsbad, CA). The chest was closed and the animals were allowed to recover. Subsequently, the animals were injected Buprinex (0.05 mL subcutaneously) during the first 24 hours to alleviate pain and were maintained until 4 weeks before euthanasia and recovery of the heart tissue samples.
The animals (n=8 in groups 1, 2, and 3 each and n=11 in group 4) were anesthetized, and transthoracic echocardiography was performed as detailed in the Online Supplement.
Histological and Immunohistological Studies
The animals were euthanized at 7 days and 4 weeks after heart function studies. The heart tissue samples were removed and used for histological and immunohistological studies as described earlier.12
Overexpression of Stemness Factors and SMs Reprogramming
The SMs were characterized for purity by flow cytometry for desmin expression (On-Line Figure II, A and B) and by immunocytochemistry for desmin and vimentin expression (Online Figure II, D) and RT-PCR for MyoD and vimentin expression (Online Figure II, E). We successfully reprogrammed NatSMs to achieve pluripotency subsequent to overexpression of 4 stemness factors Oct3/4, Klf4, Sox2, and c-Myc (Figure 1A, B1–B3). Culture of the undifferentiated SiPS on MEFs or on gelatin-coated dishes without feeder cells exhibited a typical ES-like cell morphology that appeared as compact, opaque round clusters with well-defined margins in the undifferentiated state and expressed Oct3/4 promoter driven GFP (Figure 1, B1–B3). On the other hand, the nonreprogrammed NatSMs with elongated spindle shape retained their morphological characteristics in culture for 20 days and did not express Oct3/4 promoter-driven GFP, which indicated their nontransformed status (Figure 1C and 1D). Subcellular characteristics included a large nucleus/cytoplasmic ratio and scanty cytoplasmic contents in SiPS in comparison with the NatSMs (Figure 1E through 1G). Unlike NatSMs, SiPS endogenously overexpressed Oct4, Sox2, cMyc, Klf4, and Nanog besides a panel of other pluripotency gene markers (Figure 1H, lanes 3 to 4), which was comparable with mouse ES cells (Figure 1H, lane 5). RT-PCR was performed to observe changes in exogenous and endogenous gene expression of stemness factors in SMs and SiPS (Online Figure III). Fluorescence immunostaining revealed that reprogramming induced strong positive expression of the undifferentiated ES cell markers, including SSEA-1 and higher alkaline phosphatase, which were absent in NatSMs (Online Figure I, A and B). Karyotyping revealed that more than 85% of the cells were euploid and without any chromosomal abnormalities, indicating that the clones used in the study possessed normal genetic makeup. Subcutaneous injection of SiPS formed teratomas in all the 3 immunodeficient hosts, which showed typical 3 germ layer characteristics (Online Figure I, C).
In Vitro Predifferentiation of Cardiac Progenitors From SiPS for Transplantation
Of the 2 murine SiPS clones, ie, Raf1 and Raf2, characterized in vitro for pluripotency, Raf1 was differentiated by a standard EB-based differentiation protocol. Between days 3 and 5 in differentiation culture medium, the floating EBs were transferred to adherent culture conditions in which the cells were cultured for 5 to 6 days. By day 10 after initiation of differentiation, few spontaneously beating areas were observed that continued to increase and expand with longer time maintenance of the culture (Online Movie I). Such synchronous contractile activity in SiPS-CMs was observed for up to 3 to 4 weeks. Profiling of cardiac specific genes was performed for characterization of the spontaneously beating EBs (Figure 2). We observed multifold increase in cardiac-specific genes expression in 10-day-old beating EBs, including maturation markers α-MHC, β-MHC, and cardiac troponin-I in comparison with SiPS and NatSMs (Figure 2A and Online Figure IV). Mouse heart was used as a positive control. The cardiac-specific molecular changes during differentiation of SiPS were confirmed by fluorescence immunostaining of SiPS and SiPS-CMs (Figure 2B). A higher propensity of SiPS-CMs positive for Nkx2.5, myosin heavy chain, cardiac troponin-I, cardiac actin, and gap-junction proteins N-cadherin and connexin-43. Incidentally, the level of pluripotency markers Oct3/4, Sox2, and Nanog concomitantly declined in SiPS during differentiation to become SiPS-CMs (Figure 2C, Online Figure V). RT-PCR was also performed to observe gene expression changes in endogenous and exogenous Oct3/4 and Sox2 in SiPS-CMs in comparison with SiPS (Online Figure VI).
Transmission electron microscopy revealed ultrastructural features of the developing cardiomyocytes in SiPS-CMs with fully developed sarcomeres and ribosomes (Figure 3, A through C). Tight junctions between adjacent SiPS-CMs were also observed in the spontaneous beating regions in the culture (indicated by red arrows;Figure 3C).
Prevention/Reduction of Tumorgenicity by Predifferentiated Cardiac Progenitors
As published in our first report on teratogenicity of SiPS in which 6 of the 16 SiPS-transplanted animals developed cardiac tumors,13 no tumor formation was observed in any other group included in the present study. Figure 4C is a representative echocardiograph of an animal heart that developed cardiac tumor in the LV 4 weeks after SiPS transplantation. Histological studies confirmed that these tumors consisted of cells from the 3 germ layers.13 Transmission electron microscopy revealed regions with myogenic differentiation identified on the basis of their typical striated muscle fiberlike structure with sarcomeric organization (Figure 4D). Moreover, comparative miRNA profile revealed that cardiomyocytes isolated from SiPS expressed tumor suppressive miRs 125b, 16, 199a, 214, 26a, 200b, and 200c in abundance when compared with SiPS (Figure 5). These results suggest that directed differentiation of iPS cells prior to transplantation is highly desirable to prevent tumor formation.
Histological Benefits of SiPS and Their Derivatives
Histochemical studies on the animal hearts from various treatment groups (n=4 per group), 4 weeks after their respective treatment, were performed on at least 2 middle slices of the heart. We observed extensive myocyte damage in the LV, which was replaced by scar tissue and thinning of the LV wall was reduced in all the treatment groups (Figure 4). However, all the cell treatment groups had significantly attenuated infarct size in comparison with DMEM-injected controls (41.3%±1.3%). However, infarct size between cell treatment groups did not show significant difference despite the fact that SiPS-CMs– treated animal hearts had the smallest infarct size (23.9%±3.1%) in comparison with SMs (25.9±1.8) and SiPS treated (26±3.3). Architectural analysis of hematoxylin-eosin–stained cardiac tissues had extensive presence of islands of myofibers in the infarcted hearts of the cell-treated groups (Figure 4). Four weeks after cell transplantation, Q-dot-labeled cells were observed in the infarct and peri-infarct areas in the LV (Figure 6A). Immunohistochemistry for cardiac troponin-I and Cx-43 was performed to determine the fate and integration of the transplanted cells in the heart. Colocalization of Q-dots (red fluorescence) with cardiac troponin-I (green fluorescence) in the infarct and peri-infarct regions in the SiPS-CMs–transplanted animal hearts indicated their myogenic differentiation (Figure 6, B through D), which formed gap junctions with the host myocytes (Figure 6, E and F).
Heart Function Studies
Permanent LAD coronary artery ligation led to significant deterioration in the indices of LV contractile function, including LVEF and LVFS in comparison with the baseline values (n=4; 74.2±2.6; 36.7%±2.2%, respectively). Transplantation of NatSMs, SiPS, and SiPS-CMs significantly preserved the global pump function of the infarcted myocardium (Figure 7). In comparison with the DMEM-treated controls (36.3%±1.5%; 15.35±1, P<0.001), LVEF and LVFS were significantly higher in NatSMs-transplanted animal hearts (48.04%±1.5%; 20.4±1.6), SiPS-transplanted hearts (46.7±1.7; 19.9±1.3) and SiPS-CMs-transplanted hearts (54.7±2; 24.1±2), respectively. Intergroup analysis showed that in comparison with DMEM treatment, both LVEF and LVFS significantly improved in cell-transplanted groups (DMEM- versus cell-transplanted groups, P<0.05). However, LVEF and LVFS showed significant improvement in SiPS-CMs-treated group in comparison with other cell-transplanted hearts. The pathological remodeling of LV in the cell-transplanted groups was also significantly reduced as shown by LV chamber dimensions during systole (LVDs) and diastole (LVDd). In comparison with the chamber dimensions during systole (4.4±0.2) and diastole (5.69±0.3) in the DMEM-treated group, both LVDs and LVDd were significantly preserved following intervention with NatSMs (3.7±0.1 and 4.7±0.1), SiPS (3.9±0.2 and 4.9±0.24), and SiPS-CMs (3.4±0.2 and 4.4±0.1), respectively.
The breakthrough discovery that somatic cells could be reprogrammed to pluripotent status has generated immense interest in pluripotent stem cells because of their vast therapeutic applications. Thus, the prospect of using iPS cells has added new dimensions to stem cell–based therapy. We report reprogramming of mouse SMs and their application for cardiac repair following myocardial infarction in comparison with SiPS and NatSMs. Our results avidly showed the superiority of allogenic SiPS-CMs in terms of safety and regenerative capacity in an immunocompetent mouse model of acute myocardial infarction. The major findings of the study are that (1) SMs can be easily reprogrammed to develop iPS cells; (2) undifferentiated iPS cells are tumorgenic in immunocompetent hosts; (3) directed differentiation of iPS cells to derive cardiac progenitors circumvent their tumorgenicity following transplantation in immunocompetent recipients; and (4) we provide a comparative miR expression profile in NatSMs, SiPS, and SiPS-CMs that essentially determined the pluripotent status of SiPS during reprogramming of SMs and showed an upregulated expression of tumor-suppressive miRs in differentiated cardiac cells.
The rationale to reprogram SMs was based on our results that SMs endogenously expressed 3 (ie, Sox2, cMyc, and Klf4) of the Yamanaka's quartet of transcription factors required for reprogramming,6 thus making these cells an easier choice for reprogramming than were the terminally differentiated fibroblasts. It is highly likely that reprogramming SMs would require fewer factors.14 Second, it is anticipated that iPS cells carry forward the epigenetic memory of the somatic cells of their origin.15 Therefore, SMs that possess inherent myogenic potential would be a better choice for reprogramming than would the nonmyogenic fibroblasts. Despite the presence of 3 of the 4 required stemness factors, the use of Oct4 alone did not induce pluripotency in SMs. It appears that there is an optimum threshold level of expression for each of these factors that is required to ensure reprogramming of SMs. A recent study has shown that SMs can be reprogrammed using Yamanaka's quartet of stemness factors with simultaneous suppression of MyoD in the reprogrammed cells, which substantiates our results.15 However, the authors did not show their cardiac differentiation capability in vitro as well as in an experimental animal model. In an interesting study, neurospheres were obtained from mouse iPS cell lines derived from embryonic fibroblasts, adult tail-tip fibroblasts, hepatocytes, and stomach epithelial cells16 and evaluated the teratogenicity of the secondary neurospheres. Interestingly, iPS cells of different tissue origins differed in their teratogenicity, which also correlated well with the presence of undifferentiated iPS cells in the neurospheres. These data support our notion that certain somatic cell types may be superior choices for reprogramming and would also influence the subsequent differentiation potential of their derived iPS cells. Another important consideration in the selection of SMs for reprogramming was that the choice of cell may impact the tumorgenicity of their derived iPS cells. Moreover, SMs have been extensively studied for myocardial repair in both experimental animal models and human patients.17,18 However, their lack of functional integration postengraftment in the heart and the issues of arrhythmogenicity have marred their clinical applicability.19 We believe that reprogrammed SMs would provide an excellent autologous source of cells for transplantation.
Since inception of the iPS cell technology, in vivo cardiomyogenic potential of iPS cells has been mostly studied after their blastocyst injection.20,21 The injected iPS cells differentiated into mature cardiomyocytes in the chimeric heart. These data were well supported by in vitro experimental studies that examined the cardiomyogenic potential of ES cells.20,22 Molecular studies demonstrated upregulation of mesodermal gene and protein markers of cardiac differentiation, which was confirmed by fluorescence immunostaining and ultrastructural studies. Similar to ES cells, iPS cells can differentiate into functionally competent cardiomyocytes,23 however, very few studies showed that iPS cells regenerated the infarcted myocardium after transplantation. Terzic et al have reported improved global cardiac function in iPS cell–treated mice, however, without substantiating cardiogenesis in the infarcted mice heart with immunohistological evidence.8 Given the tumorgenic nature of pluripotent stem cells, it is important to ensure that the transplantation of SiPS is free of undifferentiated cells,24 which are potential contributors of tumorgenicity of iPS cells.16 Even the use of isogenic animals for transplantation of SiPS failed to curtail their teratogenic characteristics in the heart. Isolation of specific subtypes of SiPS with no oncogenic tendency will also help to curtail tumorgenicity of SiPS.
In view of difficulties in using iPS cell–based cell therapy, we hypothesized that predifferentiation or guided differentiation of SiPS would be a safer and effective approach for transplantation. We therefore opted to transplant 8- to 10-day-old EBs that contained developing and beating myocytes. We observed increased myogenesis in SiPS-CMs transplanted hearts and, more important, without incidence of tumor formation as against significantly higher number of animals (n=4) developing tumors in SiPS transplanted hearts. Because the number of cells transplanted in group 3 and group 4 were similar, contribution of residual SiPS population toward improved cardiac function would be marginal. Strategies to enhance the purity of SiPS-CMs and to exclude the presence of undifferentiated SiPS are therefore warranted. Directed differentiation protocols will require optimal pretreatment of SiPS with specific cytokines, growth factors, or small molecules rather than the use of spontaneous differentiation, which may lack reproducibility.
Our study provides the first ever miR expression profiling of SMs during the process of reprogramming and differentiation. The miRs are global regulators of stem cell functions, including their differentiation capacity.25 Significant changes were noticed in the expression pattern of miR let-7, miR-200, and miR-290 families that largely varied with the differentiation status of the cells. Upregulation of let-7 family of miRs is associated with differentiation status rather than type of the cell.26 We observed that let family of miRs including let7 a-g, i, and miR-98 were down-regulated in SiPS in comparison with NatSMs. Nevertheless, there was an obvious increase in their expression during myogenic differentiation of SiPS. The 5-member miR-200 family (miR-200a-c, miR-141, and miR-429) also showed a similar trend. Of these members, miR-200a-c were not detected in either NatSMs or SiPS; however, their expression increased in SiPS-CMs, which indicated the incomplete differentiation status of SMs.26 Besides these 2 important miR families, SiPS also expressed other signature ES cell miRs—including miR-290, 292, and 293—,that are not only regulated by pluripotency genes Oct4, Nanog and Sox2 but also target these genes in “incoherent feed-forward loops.” These data reflected similarity of miR expression profiles between SiPS and ES cells. Similarly, some other miRs with significantly altered expression during transition of SMs to SiPS and then onward differentiation into cardiomyocytes included miR-23a and 23b (regulated by cMyc),27 miR-214 (part of regulatory circuit controlling differentiation and cell fate decision),28 and miR-21, a known suppressor of Nanog, Sox2, and Oct4,25 that showed substantial increase following cardiomyogenic differentiation of SiPS. The miR-24 a ubiquitously expressed miRNA, has an antiproliferative effect independent of p53 function29 and was inhibited in SiPS in comparison with SMs and SiPS-CMs. Moreover, miR-26a is a muscle- specific miR that is up-regulated during myogenesis30 and was up-regulated both in SMs and in SiPS-CMs in comparison with the SiPS. Let-7, miR-125b, miR-16, miR-199a, miR-214, miR-26a, miR-200b, and miR-200c are among the tumor-suppressive miRs that are significantly up-regulated in SiPS-CM.31–33 All these data clearly reflect not only the similarity between miR expression profile of ES cells and SiPS during reprogramming of SMs but also up-regulation of tumor-suppressive miRs in SiPS-CMs in comparison with SiPS.
In conclusion, the study results indicate SMs as better candidates for reprogramming to pluripotency because of their inherent expression of 3 of the 4 stemness-determining factors. Although the study used conventional retroviral vector-based ectopic expression of 4 stemness factors, intrinsic expression of 3 of the required 4 stemness factors raises the possibility of their reprogramming with a smaller number of factors or even by treatment with nonviral methods for clinical applications. Transplantation of cardiac progenitors predifferentiated from SiPS was optimal for tumor-free cardiogenesis with resultant structural and functional recovery in an immunocompetent animal model of acute myocardial infarction. Given that tumorgenesis is a time-dependent process, it would be prudent to carry out longer-term studies to assess tumorgenic potential of SiPS-CMs besides designing more systematic studies for guided differentiation of SiPS for transplantation.
Source of Funding
This work was supported by National Institutes of Health (NIH) grants R37-HL074272, HL080686, HL107957, and HL087246 (to M.A.) and HL087288, HL089535, and HL106190 (to K.H.H.).
Nothing to disclose.
In April 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.
Nonstandard Abbreviations and Acronyms
- skeletal myoblasts
- embryonic stem
- induced pluirpotent stem
- skeletal myoblast derived iPS cells
- embryoid bodies
- cardiomyocytes derived from the 10-day-old spontaneously beating embryoid bodies obtained from SiPS
- green fluorescent protein
- Dulbecco's modified eagle medium
- octamer-binding transcription factor 4
- sex determining region Y-box 2
- Kruppel-like factor 4
- cellular-myelocytomatosis proto-oncoprotein gene
- mouse embryonic fibroblasts
- antistage specific embryonic antigen-1 antibody
- connexin 43
- myogenic differentiation 1
- native skeletal myoblasts
- quantum dots
- left ventricular chamber dimensions during systole
- left ventricular chamber dimensions during diastole
- left ventricular ejection fraction
- left ventricular fractional shortening
- Received December 30, 2010.
- Revision received April 25, 2011.
- Accepted May 4, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is known?
Committed skeletal muscle progenitor cells (myoblasts) can be reprogrammed into pluripotent stem cells that are similar to embryonic stem cells.
Skeletal myoblast–derived induced pluripotent stem cells (SiPS) are potential candidates for the regeneration of ischemic myocardium.
SiPS transplanted into the ischemic myocardium have been reported to differentiate into cells of all 3 germ layers, leading to tumor formation in the heart.
What New Information Does This Article Contribute?
Predifferentiation of SiPS into developing cardiomyocytes before transplantation is important for regeneration of infarcted myocardium with minimal risk of myocardial tumor formation.
Cardiac progenitors derived from SiPS are an excellent source of cells for cardiac regeneration. Transplantation of these cells was found to reduce infarct size and improve heart function in a mouse model of acute myocardial infarction.
In addition to known pluripotency-related miRs such as Let-7 family miRs and 290 cluster miRs, this study also reveals that other miRs—such as 125b, 16, 199, 214, 26a, and 200a/b, which are tumor-suppressive miRs—are expressed in differentiated cardiomyocytes. These miRs can be used as tumorigenic biomarkers of iPS cells–derived progenitors.
A major goal of cardiac stem cell research is to identify the ideal type of cell for cardiac regeneration. Skeletal myoblasts have been used in clinical trials involving cardiac ischemia, albeit myoblast transplantation is reported to cause arrhythmias. We have recently reported that myoblasts can be reprogrammed to induce pluripotent stem cells, and their direct transplantation is associated with a high risk of tumor formation even in an immunocompetent host. In this study we differentiated myoblast-derived IPS into cardiomyocytes (SiPS-CM) prior to transplantation to assess their potential to regenerate the infarcted myocardium. Our in vivo results in a mouse model of myocardial injury indicate that differentiation before transplantation reduces the risk of tumor formation while simultaneously improving heart function and reducing infarct size. Our results suggest that skeletal myoblasts may be better candidates for reprogramming using novel nonviral approaches because they show a modest expression of 3 of the 4 stemness genes at baseline. By comparing the miRNA profile of committed myoblasts and cardiomyocytes with the miRNA profile of SiPS, this study highlights the miRs that are specifically regulated under pluripotent state. These pluripotency-specific miRs are ideal candidates for nonviral miRNA reprogramming of differentiated cells. Moreover, as pluripotency and tumorigenicity are closely regulated, this study could be helpful in identifying potential miRNA biomarkers for turmor-free cardiac regeneration.