Therapeutic Transdifferentiation
Authors: John P. Cooke
Journal: Circulation Research (2013)

HomeCirculation ResearchVol. 112, No. 5Therapeutic Transdifferentiation Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBTherapeutic TransdifferentiationA Novel Approach for Vascular Disease John P. Cooke John P. CookeJohn P. Cooke From the Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA. Originally published1 Mar 2013https://doi.org/10.1161/CIRCRESAHA.113.301053Circulation Research. 2013;112:748–750Direct Reprogramming of Fibroblasts into Endothelial CellsCapable of Angiogenesis and Re-endothelialization inTissue-engineered VesselsMargariti et alProc Natl Acad Sci USA. 2012;109:13793–13798.Emerging evidence indicates that overexpression of a few master regulators can dramatically alter cell type (transdifferentiation), in a process called direct reprogramming. The recent reprogramming of human fibroblasts into cardiomyocytes and endothelial cells (ECs) augurs a new era in cardiovascular medicine by increasing the feasibility of cardiovascular regenerative therapy.Reprogramming Fibroblasts to ECsIn a recent seminal article, Margariti et al1 described their success in reprogramming human fibroblasts into ECs. These induced ECs had all of the phenotypic characteristics of genuine ECs. They stained for endothelial markers such as CD31, vascular endothelial cadherin, endothelial nitric oxide synthase, and von Willebrand factor. Moreover, they exhibited endothelial behaviors such as network formation in Matrigel and capillary formation in vivo. Furthermore, injection of these cells into the ischemic murine hindlimb improved perfusion, in association with an increase in capillary density. Finally, the cells were capable of contributing to re-endothelialization in tissue-engineered vessels.Nobel Prize for Induced Pluripotent Stem CellsAn important precedent for their accomplishment is the work of Shinya Yamanaka, who won the 2012 Nobel Prize in Physiology or Medicine. Dr Yamanaka discovered that the forced expression of 4 master regulators (the genes encoding the transcriptional factors Oct4, Sox2, Klf4, and cMyc) could generate induced pluripotent stem cells (iPSCs).2,3 When fibroblasts are transfected by viral vectors or RNA4,5 encoding these genes, a small percentage of the transfected cells will gradually transform (over a period of ≈2–4 weeks) into pluripotent stem cells. These iPSCs are capable of becoming endoderm, ectoderm, or mesoderm, and any of the somatic cell lineages can potentially be derived. The mechanisms by which the master regulators activate the core pluripotency network and induce the genetic and epigenetic changes required for pluripotency are incompletely elucidated and the subject of frenetic activity in the stem cell field. As a result, new insights are occurring with regularity, and our understanding of the reprogramming process is deepening. Despite the nascent state of our knowledge, iPSCs appear to have great value as a scientific tool. They have been differentiated into somatic cells for studies of pathobiology and hold great promise in the screening of small molecules for potential therapies.6,7Obstacles to iPSC TherapiesHowever, the use of iPSCs (or more likely cells derived from them) for regenerative medicine is farther off on the horizon. Concerns have been raised about the epigenetic differences between iPSCs and embryonic stem cells.8 The iPSC-derived cells may retain an epigenetic memory of the parental cells9 that could influence their function in unanticipated ways. Furthermore, differentiation protocols to therapeutic somatic cells are empirical and inefficient. Finally, methods for purifying the therapeutic cells (and, particularly, for excluding parental pluripotent cells) need to be improved.10 It is theoretically possible that the administration of 1 pluripotent stem cell could give rise to a teratoma, given the replicative capacity of these cells. Accordingly, a method to generate the desired therapeutic cell directly, that is, from another somatic cell (direct reprogramming), is appealing because this capability would avoid the risk of administering a stray pluripotent stem cell that could become a teratoma.Direct ReprogrammingIndeed, several groups have now demonstrated the feasibility of direct reprogramming. Their work owes much to John Gurdon, who also received the 2012 Nobel Prize in Physiology or Medicine. By transferring somatic cell nuclei into an enucleated oocyte, he revealed that cytoplasmic factors in the oocyte were capable of reprogramming the somatic nucleus toward pluripotency.11 Subsequent nuclear transfer experiments by others revealed that cytoplasmic factors in one somatic cell could reprogram another, including the studies of Helen Blau, which revealed that the mammalian skeletal muscle cell could reprogram the hepatocyte nucleus toward a muscle phenotype.12 Thus, the idea was born that the forced expression of master regulators might permit a form of biological alchemy, where one somatic cell was transformed into another.This notion was the inspiration for recent work toward directed reprogramming. Typical of these studies were those of Marius Wernig, who manipulated a set of ≈20 transcriptional factors known to be important in neuronal development, overexpressing them in fibroblasts.13 Combinations of these factors were assessed, and a smaller set of 4 factors was found to be sufficient for generating neurons when transfected into murine fibroblasts. This approach has also been used to generate other somatic cells from fibroblasts, including cardiomyocytes.14,15Reprogramming Fibroblasts to ECs With the Yamanaka FactorsMargariti et al1 took a slightly different approach to reprogramming fibroblasts to ECs. Rather than a set of factors known to be involved in endothelial development, they transfected fibroblasts with the Yamanaka factors before placing them into endothelial differentiation medium. They reasoned that, after a few days in culture, the overexpression of the Yamanaka factors would generate partially induced pluripotent cells (PiPS), that is, cells that were incompletely reprogrammed. Indeed, PiPS cells did not form teratomas in vivo and did not express pluripotency surface markers. The PiPS were then differentiated toward ECs by prematurely replacing the stem cell media with endothelial differentiation media. Indeed, PiPS displayed the potential to differentiate into ECs (PiPS-EC) when they were exposed to the appropriate culture conditions.An insight into the nature of this transdifferentiation came from their observation that SET translocation (myeloid leukemia-associated) similar protein was induced during the reprogramming process. Notably, when PiPS cells were treated with vascular endothelial growth factor, SET similar protein translocated to the cell nucleus, directly bound to the vascular endothelial cadherin promoter, increasing vascular endothelial cadherin expression levels and EC differentiation. Thus, one of the key factors in the endothelial culture medium was shown to contribute to PiPS differentiation to endothelial lineage.It can be argued that Margariti et al1 did not actually induce direct reprogramming of fibroblasts to ECs. For direct reprogramming, most others have used an empirically determined combination of genes encoding transcriptional factors involved in the developmental program of their chosen lineage. By contrast, Margariti et al1 used the Yamanaka factors to induce an intermediate state of partial reprogramming. Complete reprogramming occurs several weeks after exposure to retroviral vectors carrying the Yamanaka factors, under the influence of culture conditions favoring pluripotency. Margariti et al1 removed the cells from stem cell culture conditions 4 days after exposure to the Yamanaka factors (well before complete reprogramming) and placed them into culture conditions favoring endothelial lineage. It is possible that the partially reprogrammed cells included precursor cells of mesodermal lineage that could respond to the endothelial culture conditions, but the precursor cells were not fully characterized. [Of note, Ding and colleagues16,17 have used a similar approach to reprogram fibroblasts to neurons or to cardiomyocytes, suggesting that partially reprogrammed cells are a heterogenous lot and/or they have sufficient plasticity to differentiate toward different lineages given the right culture conditions]. Single-cell transcriptional studies and lineage tracing would have been helpful in characterizing the population of partially reprogrammed cells.Unanswered QuestionsThat being said, in most of the reported studies of direct reprogramming there has not been a comprehensive analysis of the transitional cells that may be created during the process of transdifferentiation. Furthermore, the epigenetic mechanisms and transcriptional programs underlying the process of transdifferentiation to any cell type are incompletely understood. One of the most perplexing questions is how a handful of transcriptional factors (and, in some cases, a single transcription factor!)18 can induce direct reprogramming of a somatic cell to one of a different germ layer (eg, fibroblast to neuron). This is particularly puzzling when the overexpressed gene does not encode a pioneering transcriptional factor, that is, one that does not itself initiate the transcriptional complex that includes other cofactors and epigenetic modifiers.One potential explanation is that the viral vectors or modified mRNA (mmRNA) that encode the transcriptional factors are having additional effects that are important in reprogramming. Recently, we have discovered that innate immune signaling plays a critical role in reprogramming somatic cells to pluripotency.19 The activation of the toll-like receptor TLR3 by viral vectors or mmRNA triggers a signaling cascade that results in global changes in epigenetic modifiers. We observed that several of the histone deacetylase family members are dramatically downregulated, whereas some histone acetyl transferase genes are upregulated, effects that would favor an open chromatin configuration. We found that knockdown of innate immunity signaling in human fibroblasts dramatically reduced their susceptibility to reprogramming by retroviral vectors or mmRNA encoding the Yamanaka factors. By contrast, activation of TLR3 by an irrelevant virus, or by poly I:C, markedly increased the efficiency of reprogramming using the Yamanaka factors in the form of cell-permeant peptides. Thus, activation of innate immunity by viral vectors or mmRNA increases epigenetic plasticity to permit cell transformation, a process that we have termed "transflammation."Transflammation and ReprogrammingIs it possible that transflammation also plays a role in the direct reprogramming using viral vectors or mmRNA? Certainly, the induction of an open chromatin configuration would make it easier to explain how a single transcriptional factor might reprogram a somatic cell, together with extrinsic cues from the media and/or extracellular matrix. In this regard, there is insufficient credit attributed to culture conditions that are used to favor growth of the desired cell type. It is likely that these culture conditions also influence differentiation to the target somatic cell. Preliminary studies in our laboratory support the notion that activation of innate immunity and external signals provided by the media and extracellular matrix may be powerful influences on direct differentiation.The Promise and Peril of Direct ReprogrammingIn any event, direct reprogramming to the desired cell type may have greater potential for regenerative medicine applications. Although human ECs derived from induced pluripotent stem cells (iPSC-EC) are functionally effective in forming capillaries and improving perfusion in vivo,20 generation of pluripotency followed by differentiation to the desired cell type takes months, and there remain concerns about teratoma formation. Direct reprogramming would avoid these concerns (although the fidelity of reprogramming requires confirmation). What is most exciting about direct reprogramming is the possibility that it might be achievable in patients in vivo. Such an approach, if accomplished with small molecules, would avoid the more complex approach of cell delivery. Indeed, recent work reveals that, in the ischemic murine myocardium, intramyocardial injection of mmRNA encoding 4 transcriptional factors is sufficient to induce direct reprogramming of cardiac fibroblasts to cardiac myocytes.14 Although the frequency of the conversion to myocytes was quite low, this exciting result provides compelling support to develop more efficient transdifferentiation techniques for clinical trial.The clinical applications for direct reprogramming of fibroblasts to ECs are numerous. For example, in ischemic injury such as myocardial infarction, one might convert the cardiac fibroblasts that are migrating and proliferating into the ischemic region into ECs. The intent would be to generate a microvasculature that could provide the nutrition and niche for reparative resident cells that could reconstitute the myocardium. As another example, it is possible that, in diseases characterized by fibrosis and loss of the microvasculature (eg, scleroderma), reprogramming of fibroblasts to ECs could reverse the scarring process.However, for the promise of this regenerative therapy to be achieved, greater efficiency of the reprogramming process is required. Furthermore, the fidelity of the reprogramming process must be assured (eg, the induced cells should ideally manifest the same transcriptional and epigenetic profile). Moreover, the integration of the reprogrammed cells within the architecture of the tissue must support organ function (eg, induced cardiomyocytes would need to form a functional syncytium with the native cardiomyocytes and not generate an arrhythmic focus; induced ECs would need to form a functional microvasculature with normal arteriovenous communications and lymphatic vessels). Potential adverse effects of direct reprogramming in vivo could include inappropriate differentiation into other cell types (eg, hamartoma) or even tumor cells. In addition, induced cells could be dysfunctional (eg, induced ECs could express more adhesion molecules and promote thrombosis or excessive inflammation). Ideally, the direct reprogramming therapy should use small molecules that can target the affected tissue and avoid gene or cell therapies that raise additional technical and regulatory obstacles. To conclude, the road to novel regenerative therapy using direct reprogramming will be long and arduous, but the journey has begun.AcknowledgmentsThis work was supported by grants to J.P.C. from the National Institutes of Health (U01HL100397, RC2HL103400, and 1UM1 HL113456).DisclosuresNone.FootnotesThe opinions expressed in this Commentary are not necessarily those of the editors or of the American Heart Association.Commentaries serve as a forum in which experts highlight and discuss articles (published here and elsewhere) that the editors of Circulation Research feel are of particular significance to cardiovascular medicine.Commentaries are edited by Aruni Bhatnagar & Ali J. Marian.Correspondence to John P. Cooke, MD, PhD, Cardiovascular Medicine, 300 Pasteur Drive, Stanford University School of Medicine, Stanford, CA 94305. E-mail: [email protected]References1. Margariti A, Winkler B, Karamariti E, Zampetaki A, Tsai TN, Baban D, Ragoussis J, Huang Y, Han JD, Zeng L, Hu Y, Xu Q. 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Hywood J, Sadeghipour S, Clayton Z, Yuan J, Stubbs C, Wong J, Cooke J, Patel S and Caporali A (2021) Induced endothelial cells from peripheral arterial disease patients and neonatal fibroblasts have comparable angiogenic properties, PLOS ONE, 10.1371/journal.pone.0255075, 16:8, (e0255075) Villarreal-Leal R, Cooke J and Corradetti B (2021) Biomimetic and immunomodulatory therapeutics as an alternative to natural exosomes for vascular and cardiac applications, Nanomedicine: Nanotechnology, Biology and Medicine, 10.1016/j.nano.2021.102385, 35, (102385), Online publication date: 1-Jul-2021. Annex B and Cooke J (2021) New Directions in Therapeutic Angiogenesis and Arteriogenesis in Peripheral Arterial Disease, Circulation Research, 128:12, (1944-1957), Online publication date: 11-Jun-2021.Meng S, Lv J, Chanda P, Owusu I, Chen K and Cooke J (2020) Reservoir of Fibroblasts Promotes Recovery From Limb Ischemia, Circulation, 142:17, (1647-1662), Online publication date: 27-Oct-2020.Cooke J and Meng S (2020) Vascular Regeneration in Peripheral Artery Disease, Arteriosclerosis, Thrombosis, and Vascular Biology, 40:7, (1627-1634), Online publication date: 1-Jul-2020. Tian G, Zhou J, Liu X and Huang Y (2019) Mechanoresponse of stem cells for vascular repair, World Journal of Stem Cells, 10.4252/wjsc.v11.i12.1104, 11:12, (1104-1114), Online publication date: 26-Dec-2019. Cooke J (2019) Inflammation and Its Role in Regeneration and Repair, Circulation Research, 124:8, (1166-1168), Online publication date: 12-Apr-2019. Clayton Z, Yuen G, Sadeghipour S, Hywood J, Wong J, Huang N, Ng M, Cooke J and Patel S (2017) A comparison of the pro-angiogenic potential of human induced pluripotent stem cell derived endothelial cells and induced endothelial cells in a murine model of peripheral arterial disease, International Journal of Cardiology, 10.1016/j.ijcard.2017.01.125, 234, (81-89), Online publication date: 1-May-2017. Sayed N, Ospino F, Himmati F, Lee J, Chanda P, Mocarski E and Cooke J (2017) Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming, Stem Cells, 10.1002/stem.2607, 35:5, (1197-1207), Online publication date: 1-May-2017. Cooke J, Meng S, Wong W, Sayed N, Ospino F, Lee J, Jha A, Dexheimer P and Aronow B (2015) Response to Letter Regarding Article "Transdifferentiation of Human Fibroblasts to Endothelial Cells: Role of Innate Immunity", Circulation, 10.1161/CIRCULATIONAHA.115.016792, 132:15, Online publication date: 13-Oct-2015. 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Anchan R, Gerami-Naini B, Lindsey J, Ho J, Kiezun A, Lipskind S, Ng N, LiCausi J, Kim C, Brezina P, Tuschl T, Maas R, Kearns W, Williams Z and Robson C (2015) Efficient Differentiation of Steroidogenic and Germ-Like Cells from Epigenetically-Related iPSCs Derived from Ovarian Granulosa Cells, PLOS ONE, 10.1371/journal.pone.0119275, 10:3, (e0119275) Cooke J, Sayed N, Lee J and Wong W (2014) Innate immunity and epigenetic plasticity in cellular reprogramming, Current Opinion in Genetics & Development, 10.1016/j.gde.2014.11.002, 28, (89-91), Online publication date: 1-Oct-2014. March 1, 2013Vol 112, Issue 5 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.113.301053PMID: 23449543 Originally publishedMarch 1, 2013 PDF download Advertisement