EDITORIAL COMMENT
A Big Promise From the Very SmallIdentification of Circulating Embryonic Stem-Like Pluripotent Cells in Patients With Acute Myocardial Infarction*
Douglas W. Losordo, MD, FACC* and
Raj Kishore, PhD
Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
* Reprint requests and correspondence: Dr. Douglas W. Losordo, Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Tarry 14-725, 303 East Chicago Avenue, Chicago, Illinois 60611 (Email: d-losordo{at}northwestern.edu).
Key Words: stem cell • progenitor cell • regenerative medicine • bone marrow
The available evidence showing improvement in myocardial function after transplantation of autologous bone marrow (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 acute myocardial infarction (MI) patients (1). Additionally, risk factors for coronary artery disease are reported to be associated with a reduced number of and impaired functional activity of endothelial progenitor cells (EPCs) in the peripheral blood of patients (2,3). Heterogeneity of BM-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 (ESCs) is therefore argued to be considered for their potential application in regenerative medicine. Although ESCs 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 ESCs for cardiac repair in humans. Moreover, successful generation of patient-specific ESCs by therapeutic cloning is yet to be achieved.
Lately, oocyte-independent generation of ESCs, also termed as induced pluripotent cells (iPS), by epigenetic reprogramming of adult somatic cells has generated significant enthusiasm. These studies, including those from 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-ESC fusion (6,7) strategy, although excellent for mechanistic studies, retains certain drawbacks that are associated with oocyte-dependent therapeutic cloning. First, the 2 cells used to generate hybrid cells are not derived from an autologous source; second, the efficiency of fusion remains low (1 to 3 in 1,000); third, the genetic stability of heterokaryon hybrids remains to be established: one will have to devise the technological innovations to delete the additional set of chromosomes; and finally, the efficacy of reprogrammed cells to retain the ESC-like properties if the ESC-derived nucleus is removed remains to be elucidated. Recently, a major breakthrough was reported whereby forced expression of transcription factors Oct-4, Sox2, c-Myc, and Klf4 were shown to induce pluripotency in primary mouse fibroblasts (8) and in human fibroblasts using either Oct-4, Sox2, c-myc, and Klf4 combination (9) or Oct-4, Nanog, Sox2, and lin28 combination (10). Although these studies did provide the evidence that overexpression of these ESC-specific genes leads to the derivation of embryonic stem-like cells from primary human fibroblasts, several critical questions were not answered by these studies. First, it seems that the requirement of these factors or their combination is varied, indicating that some of these factors may be dispensable. Second, because 4 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 fibroblasts utilizing murine ESC-free extracts. Like the other 2 types of iPS cells discussed previously, these cells can differentiate into cells representative of all 3 germ layers and significantly enhance post-acute MI cardiac function and myogenesis when transplanted into a mouse acute MI 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-acute MI myocardial repair would therefore be one that is autologous and yet possesses ESC-like plasticity for differentiation. In this regard, the study by Wojakowski et al. (12), published in this issue of the Journal, is of considerable interest. The investigators report the identification of a subset of nonhematopoetic, BM-derived cells that express a number of ESC-specific transcripts and that are mobilized into peripheral blood in patients after acute MI. These cells, termed very small embryonic-like stem cells (VSELs) based on their size (7 to 8 µm), 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 investigators show that VSELs are present in the circulation of both healthy subjects and patients with acute MI. Interestingly, acute MI 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 Oct-4 and Nanog as well as cardiomyocyte and endothelial lineage-specific transcripts. The current report thus builds on the extensive functional and phenotypic characterization of murine VSELs reported in several articles by the same group of investigators (13–17). The VSELs were reported by Kucia et al. (13) as a rare population of cells in the mouse BM that were positive for Sca-1 and negative for both hematopoetic lineage marker (Lin) and the pan-leukocyte marker CD45 (Sca-1+/Lin–/CD45–). Like human VSELs identified in the current report, these cells expressed a number of pluripotency-associated markers (Oct-4, Nanog, SSEA-1, Rex1) and differentiated in vitro into components of all 3 germ layers. In addition to expressing ESC-specific transcripts, these cells were reported to coexpress markers of neuronal, pancreatic, endothelial, and cardiac lineages (13–17) and acquired cardiomyocyte phenotype in vitro. Follow-up studies reported that on experimental acute MI in mice, these cells are mobilized from the BM (16), and when transplanted in murine models of acute MI, these cells improved global and regional LV function and showed evidence of myogenesis and vasculogenesis (17). The human VSELs reported in the present study were not characterized for their functional or differentiation properties, although phenotypic characterization of these cells led the investigators 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 that is distinct from the other defined populations and that 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 that need to be approached as clinical application is considered. A central scientific question pertains to the origin of these cells, that is, whether these Oct-4+ cells are functional in steady-state conditions or represent a population of dormant cells residing in BM/tissue as leftover remnants from developmental embryogenesis. Second, do these cells represent true embryonic cell behavior and potential, because as reported by the authors previously, these cells show 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 physiological stimuli such as myocardial ischemia? In this regard, one interesting observation made in the current article is worth noting. The investigators observed that cells isolated from acute MI patients 12 h after MI show a certain level of Oct-4 transcript. However, cells isolated from patients 5 days after MI showed many-fold higher expression of Oct-4 messenger ribonucleic acid. These data would indicate that there is a continuous transcription of Oct-4 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 VSELs is their rarity. Either refinement of methods to isolate larger numbers of VSELs or ways to expand them ex vivo may be required for clinical applications. Finally, as reported by Wojakowski et al. (12), 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). Because 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 examined carefully for the VSELs.
Despite these questions, which we believe will be answered in the near future, the identification of this distinct population of autologous ESC-like pluripotent cells seems quite promising. The 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.
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* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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