A Big Promise from the Very Small: Identification of Circulating ES-like Pluripotent Cells in Patients with Acute Myocardial Infarction

The available evidence demonstrating improvement in myocardial function following transplantation of autologous BM derived stem/progenitor cells, both in pre-clinical as well as in available clinical trials, remains a potent force driving discovery and clinical development simultaneously and has provided new hope for patients with debilitating heart diseases. However, certain potential limitations of autologous BM or peripheral blood derived stem/progenitor cells have been recently identified. Long-term follow up in one study revealed a “catch up” of placebo treated patients, resulting in an decrease in the initially observed advantage of BM cell infusion in AMI patients (1). Additionally, risk factors for coronary artery disease are reported to be associated with a reduced number and impaired functional activity of EPC in the peripheral blood of patients (2,3). Heterogeneity of bone marrow derived stem cells; incomplete mechanistic insights into their function, limited plasticity and trans-differentiation potential to various lineages of cells are also the subject of intense debate. Moreover, the stability of trans-differentiated cells to maintain newly acquired phenotype and the heritability of such changes remain to be defined. The inherent plasticity of embryonic stem cells (ESC) is therefore argued to be considered for their potential application in regenerative medicine. Although ESC have been used in animal studies of cardiac repair (4,5), ethical and regulatory issues as well as immunogenicity limit the potential therapeutic utility of ESC for cardiac repair in humans. Moreover, successful generation of patient-specific ESC by therapeutic cloning is yet to be achieved.

Lately, oocyte-independent generation of ESC, also termed as induced pluripotent cells (iPS), by epigenetic reprogramming of adult somatic cells has generated significant enthusiasm. These studies, including those form our laboratories, utilized 3 basic methodologies to generate ESCs from somatic cells: 1) Somatic-ESC fusion; 2) retroviral transduction of ESC-specific genes; and 3) exposure of permeabilized somatic cells to ESC cell-free extracts. The somatic-ES cell fusion (6,7) strategy, although excellent for mechanistic studies, retains certain drawbacks that are associated with oocyte-dependent therapeutic cloning. Firstly, the 2 cells used to generate hybrid cells are not derived from autologous source, secondly the efficiency of fusion remains low (1–3/1000), thirdly the genetic stability of heterokaryon hybrids remains to be established: - one will have to devise the technological innovations to delete additional set of chromosomes, and finally the efficacy of reprogrammed cell to retain the ES like properties if ES cell derived nucleus is removed remains to be elucidated. Recently, a major breakthrough was reported whereby forced expression of transcription factors Oct4, Sox2, c-Myc and Klf4 was shown to induce pluripotency in primary mouse fibroblasts (8) and in human fibroblasts using either Oct4, Sox2, c-myc and Klf4 combination (9) or Oct4, nanog, Sox2, lin28 combination (10). While these studies did provide the evidence that over-expression of these ES-specific genes leads to the derivation of ES-like cells from primary human fibroblasts, several critical questions were not answered by these studies. First, it appears, that the requirement of these factors or their combination is varied, indicating that some of these factors may be dispensable. Secondly, since four transcription factors were transduced by constitutively expressed retroviral vectors it is unclear why the cells could be induced to differentiate and whether continuous vector expression was required for the maintenance of the pluripotent state. Recently, we have reported the generation of ESC-like pluripotent cells from NIH3T3 fibrobalsts utililizing mESC cell-free extracts. Like the other 2 types of iPS cells discussed above, these cells can differentiate into cells representative of all 3 germ layers and significantly enhance post-AMI cardiac function and myogenesis when transplanted in mouse AMI model (11). Despite these exciting breakthroughs, these iPS cells are not yet ready to be translated into clinical applications.

The ideal cell type for clinical applications in post-AMI myocardial repair would therefore be one that are autologous and yet possess ESC-like plasticity for differentiation. In this regard the study by Wojakowski and colleagues (12), published in this issue of the Journal, is of considerable interest. The authors report the identification of a subset of non-hematopoetic, BM derived cells that express a number of ESC-specific transcripts and which are mobilized into peripheral blood in patients following AMI. These cells, termed Very Small Embryonic-Like Stem Cells (VSEL) based on their size (7–8μm), may potentially represent a cell type for use in the cardiac repair. They are endogenous, autologous, unmodified and pluripotent, thus bridging the gap between adult and embryonic stem cell phenotypes. The authors show that VSEL cells are present in the circulation of both healthy subjects and in patients with AMI. Interestingly, AMI significantly enhances mobilization of VSELs from the BM to the circulation. Phenotypic characterization of these cells confirmed that these cells express markers of pluripotency including Oct4 and Nanog as well as cardiomyocyte and endothelial lineage specific transcripts. The current report thus builds upon the extensive functional and phenotypic characterization of murine VSELs reported in several manuscripts by the same group of authors (13–17). The VSELs were reported by Kucia et al (13) as a rare population of cells in the mouse BM which were positive for Sca-1 and negative for both hematopoetic lineage marker (Lin) and the pan leukocyte markerCD45 (Sca-1+/Lin−/CD45−). Like human VSELs identified in the current report, these cells expressed a number of pluripotency-associated markers (Oct4, nanog, SSEA-1, Rex1) and differentiated in vitro into components of all three germ layers. In addition to expressing ESC-specific transcripts, these cells were reported to co-express markers of neuronal, pancreatic, endothelial and cardiac lineages (13–17) and acquired cardiomyocyte phenotype, in vitro. Follow up studies reported that upon experimental AMI in mice, these cells are mobilized from the BM (16) and when transplanted in murine models of AMI, these cells improved global and regional left ventricular function and showed evidence of myogenesis and vasculogenesis (17). The human VSEL reported in the present study were not characterized for their functional or differentiation properties, although phenotypic characterization of these cells led the authors to predict similar functional properties as observed for mouse VSELs. Taken together these observations in mice and in humans confirm the presence of a subset of BM stem cells which are distinct from the other defined populations and which possesses a greater differential potential for cardiac repair.

Although these observations are exciting and these cells may hold promise for regenerative medicine in general and cardiac regeneration in particular, it is not surprising that a novel discovery such as this also raises many questions which need to be approached as clinical application is considered. A central scientific question pertains to the origin of these cells i.e. whether these Oct4+ cells are functional in steady state conditions or represent a population of dormant cells residing in BM/tissue as left-over remnants from developmental embryogenesis. Secondly, do these cells represent true embryonic cell behavior and potential since as reported by the authors previously these cells display a certain degree of lineage commitment (e.g. neural, cardiac and endothelial). Third, does the pluripotent phenotype observed for these cells represent steady state function or does it represent an epigenetic phenomenon induced by the physiological stimuli such as myocardial ischemia? In this regard one interesting observation made in the current manuscript is worth noting. The authors observed that cells isolated from AMI patients 12 hours post- MI display certain level of Oct4 transcript. However, cells isolated from patients 5 days post-MI showed many-fold higher expression of Oct4 mRNA. These data would indicate that there is a continuous transcription of Oct4 likely induced by the signals originating from infracted myocardium. It would be interesting to know whether these signals include activation of epigenetic modifiers such as histone deacetylases and methylases leading to chromatin modifications and enhanced transcriptional activation of ESC-specific markers. A potential limitation of the VSEL cells is their rarity. Either refinement of methods to isolate larger number of VSELs or ways to ex vivo expand them may be required for clinical applications. Finally, as reported by the authors in this manuscript, the VSELs, despite being phenotypically reminiscent of ESCs, suffer similar drawbacks that are noted for other adult BM-derived stem cell populations: the number of these cells diminish with age and with other risk factors of heart disease (e.g. diabetes). Since the incidence of heart disease coincides with risk factors, this issue will be the subject of ongoing study as these cells are considered for clinical applications. Additionally, limitations associated with ESC therapy such as teratoma and tumor formation also need to be carefully examined for the VSEL cells.

Despite these questions, which we believe will be answered in near future, the identification of this distinct population of autologous ES-like pluripotent cells appears quite promising. VSELs could potentially provide a real therapeutic alternative to the ethically controversial and technically challenging oocyte-dependent therapeutic cloning and generation of individualized human ESCs. Future study of VSEL cells will provide additional clues regarding the transcriptional programs that drive pluripotency and differentiation moving our understanding of basic mechanisms forward and tissue regeneration closer to clinical reality. Until then it is safe to assume that these small cells hold a big promise for cardiac repair and for other applications in regenerative medicine.