Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells

Single-cell transplantation analysis revealed that the cells that had the strongest dye efflux activity ("Tip"-SP cells) and had the phenotype CD34- c-Kit+ Sca-1+ Lin- (CD34- KSL cells) exhibited very strong proliferation and multilineage differentiation capacity. Ninety-six percent of the lethally irradiated mice that received a single "Tip"-SP CD34- KSL cell showed significant donor cell engraftment for long term. These findings support the hypothesis that "Tip"-SP CD34- KSL cells represent the most primitive hematopoietic stem cells that are capable of migrating into the primary site and surviving and/or proliferating with nearly absolute efficiency. This led us to propose high marrow-seeding efficiency as a specific characteristic of primitive HSCs, in addition to their self-renewal and multipotent capacity.     

Immunophenotype and functional characteristics of human primitive CD34-negative hematopoietic stem cells: the significance of the intra-bone marrow injection

The biology of hematopoietic stem cell (HSC) is a current topic of interest which has important implications for clinical HSC transplantation as well as for the basic research of HSC. The most primitive HSCs in mammals, including mice and humans, have long been believed to be CD34 antigen (Ag)-positive (CD34(+)) cells. In fact, bone marrow (BM), peripheral blood (PB), and cord blood (CB) stem cell transplantation studies indicate that a CD34(+) subpopulation in the BM, PB, or CB can provide durable long-term donor-derived lymphohematopoietic reconstitution. Therefore, CD34 Ag was used to identify/purify immature HSCs. However, Osawa et al. reported that murine long-term lymphohematopoietic reconstituting HSCs are lineage marker-negative (Lin(-)) c-kit(+)Sca-1(+)CD34-low/negative (CD34(low/-)), which are called CD34(low/-) KSL cells. Recently, human CB-derived CD34(-) HSCs, a counterpart of murine CD34(low/-) KSL cells, were successfully identified using an intra-bone marrow injection (IBMI) method. This review will update the concept of the immunophenotype and the functional characteristics of human primitive CD34(-) HSCs. In addition, the significance of the application of the IBMI technique in clinical HSC transplantation is also discussed. Recent rapid advances in understanding the biological nature of HSCs may make it possible to fully characterize the most primitive class of human HSCs in the near future.     

Repopulating activity of ex vivo-expanded murine hematopoietic stem cells resides in the CD48-c-Kit+Sca-1+lineage marker- cell population

A better understanding of the biology of cultured hematopoietic stem cells (HSCs) is required to achieve ex vivo expansion of HSCs. In this study, clonal analysis of the surface phenotype and repopulating activity of ex vivo-expanded murine HSCs was performed. After 7 days of culture with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2, single CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells gave rise to various numbers of cells. The proportion of KSL cells decreased with increasing number of expanded cells. Transplantation studies revealed that the progeny containing a higher percentage of KSL cells tended to have enhanced repopulating potential. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture. Repopulating activity resided only in the CD48-KSL cell population, which had a relatively long intermitotic interval. Microarray analysis showed surprisingly few differences in gene expression between cultured CD48-KSL cells (cycling HSCs) and CD48+KSL cells (cycling non-HSCs) compared with freshly isolated CD34-KSL cells (quiescent HSCs), suggesting that the maintenance of stem cell activity is controlled by a relatively small number of genes. These findings should lead to a better understanding of ex vivo-expanded HSCs.     

Two different roles of purified CD45+c-Kit+Sca-1+Lin- cells after transplantation in muscles

Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)-labeled CD45+c-Kit+Sca-1+Lin- cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+PAX7+, GFP+MyoD+, and GFP+Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.     

Catecholaminergic neurotransmitters regulate migration and repopulation of immature human CD34+ cells through Wnt signaling

Catecholamines are important regulators of homeostasis, yet their functions in hematopoiesis are poorly understood. Here we report that immature human CD34+ cells dynamically expressed dopamine and beta2-adrenergic receptors, with higher expression in the primitive CD34+CD38(lo) population. The myeloid cytokines G-CSF and GM-CSF upregulated neuronal receptor expression on immature CD34+ cells. Treatment with neurotransmitters increased the motility, proliferation and colony formation of human progenitor cells, correlating with increased polarity, expression of the metalloproteinase MT1-MMP and activity of the metalloproteinase MMP-2. Treatment with catecholamines enhanced human CD34+ cell engraftment of NOD-SCID mice through Wnt signaling activation and increased cell mobilization and bone marrow Sca-1+c-Kit+Lin- cell numbers. Our results identify new functions for neurotransmitters and myeloid cytokines in the direct regulation of human and mouse progenitor cell migration and development.     

Murine side population cells contain cobblestone area-forming cell activity in mobilized blood

Primitive hematopoietic stem cells (HSCs) can be purified from murine bone marrow by sorting Hoechst 33342-effluxing side population (SP) cells. The aim of this study was to establish whether SP cells from peripheral blood contain primitive HSCs and whether this is altered in mice following mobilization. SP cells were analyzed and isolated from bone marrow and blood of mice after mobilization; the HSC content of isolated SP cells was determined through surrogate cobblestone area-forming cell (CAFC) assays. SP cells in normal blood were not found in the high Hoechst dye effluxing portion of the SP tail, did not express the stem cell markers c-Kit and CD34, and did not have measurable CAFC activity. In contrast, SP cells in mobilized blood expressed both stem cell markers, contained cells in the high dye efflux portion of the SP tail, and displayed significant day- 28 to day-35 CAFC activity with 165- to 334-fold enrichment. In comparison to mobilized blood SP cells, normal marrow SP cells contained a higher proportion of cells expressing c-Kit and CD34 and had a greater percentage of cells in the high Hoechst dye-effluxing portion of the SP tail. Analysis of SP cells in the bone marrow after mobilization revealed a decrease in the frequency of SP cells, in expression of c-Kit and Sca+ CD34(+)/CD34(-), and in day-7 to day-35 CAFC activity, consistent with mobilization into blood. We conclude that murine SP cells mobilized into blood contain primitive hematopoietic stem cell activity (day-28 to day-35 CAFC activity). This model offers a means to study the mechanisms of mobilization of primitive stem cells directly in a murine model.     

Fibroblast growth factor-1 and -2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures

In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules, stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.     

AGM区来源的基质细胞促进造血干细胞扩增的体外实验研究

目的： 探讨主动脉-性腺-中肾（aorta-gonad-mesonephros，AGM）来源的基质细胞对造血干细胞（HSC）增殖的促进作用，为探寻HSC的体外扩增方法奠定实验基础。 方法： 分别从孕11 d BALB/c小鼠胚胎AGM区及6周龄小鼠骨髓分离、培养基质细胞，流式细胞仪等对基质细胞进行鉴定；利用小鼠胚胎干细胞（ESC）向造血细胞定向分化的模型，结合高增殖潜能集落（HPP-CFC）、原始细胞集落（BL-CFC）形成实验及流式细胞仪分析CD34+、CD34+Sca-1+细胞比例，对比研究AGM及骨髓基质细胞对ESC来源的HSC的扩增作用。 结果： 小鼠AGM和骨髓基质细胞在形态及表型上基本相似，均符合基质细胞的特征。AGM和骨髓基质细胞均可促进ESC来源的HPP-CFC的形成，但AGM基质细胞还可促进ESC来源的 BL-CFC的形成；流式细胞仪检测发现：在骨髓基质细胞支持下，CD34+细胞增加了3-4倍，但CD34+/Sca-1+却无明显增加；而在AGM基质细胞支持下CD34+、CD34+Sca-1+细胞均明显增加了4-5倍。 结论： AGM基质细胞在有效扩增小鼠HSC同时，能很好地维持HSC自我更新及多向分化的潜能。     

Vaccinia virus infection modulates the hematopoietic cell compartments in the bone marrow

Successful proliferation and differentiation of hematopoietic progenitor cells in bone marrow (BM) is essential to generate all mature blood cell types, including those involved in the immune response. Although vaccinia virus (VV) is known to induce a strong immune response, the effect of VV infection on hematopoiesis remains largely unknown. Here, we showed that in vivo VV infection results in the expansion of c-Kit(hi)Sca-1+Lin- (KSL) hematopoietic stem cells. The in vivo expansion of the KSL population requires MyD88 that is a critical adaptor for Toll-like receptor-mediated signaling. Moreover, in BM of VV-infected mice, common myeloid progenitors (CMP) was decreased because of the rapid differentiation of CMP to more mature cells. However, the CMP compartment was not affected by VV infection in the absence of MyD88. The common lymphoid progenitor (CLP) cell population was increased regardless of MyD88 status, suggesting the independent regulation of CMP and CLP compartments by VV infection. VV infection also enhanced the potential of progenitors that preferentially induce the programming of dendritic cell (DC) development toward plasmacytoid DC. Therefore, the host immune response is gearing toward antiviral responses as early as at the precursor level upon VV infection.     

A novel function of interleukin-10 promoting self-renewal of hematopoietic stem cells

Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the factors regulating the process remain poorly understood. Here, we show that Interleukin-10 (IL-10), a pleiotropic immune modulating cytokine, can also play a role in regulating HSC self-renewal. First, a quantitative decrease of primitive hematopoietic cell populations, but not more matured cells, was observed in the bone marrows of IL-10 disrupted mice as determined by long-term in vitro cultures or in vivo competitive repopulation assays. In contrast, normal HSCs from 5-fluorouracil treated marrows cultured on the IL-10 secreting stroma displayed an enhanced repopulating activity compared with cells grown on control stroma, with ninefold higher numbers of donor-derived HSCs in the reconstituted recipient marrows. Moreover, limiting dilution transplantation assay demonstrated that exogenous addition of IL-10 in the stroma-free cultures of purified Lin- Sca-1+ c-kit+ cells caused three- to fourfold higher frequencies of HSCs in the 5-day short-term culture without indirect inhibitory effect of IL-10 on tumor necrosis factor-alpha or interferon-gamma secretion. Interestingly, primitive hematopoietic cells, including Lin- Sca-1+ c-kit+ or side population cells, expressed the surface receptor for IL-10, and microenvironmental production of IL-10 was sharply increased in the osteoblasts lining the trabecular regions of the radiation-stressed marrow but not in the steady-state marrows. These results show that IL-10 may be a ligand that can stimulate self-renewal of HSCs to promote their regeneration in addition to being a ligand for immune regulation. Disclosure of potential conflicts of interest is found at the end of this article.     

Characterization of a lineage-negative stem-progenitor cell population optimized for ex vivo expansion and enriched for LTC-IC

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133+ cells to represent a more primitive cell population than their CD34+ counterparts. However, both CD34+ and CD133+ cells still encompass cells at various stages of maturation, possibly impairing long-term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long-term in vivo hematopoiesis. We report here an optimized, rapid negative-isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage-negative (Lin-) cell population (0.10% +/- 0.02% MNC, n=12). This primitive Lin- cell population encompassed CD34+/- and CD133+/- HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin- cells being highly enriched for long-term culture-initiating cells when compared with both CD133+ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c-Kit ligand 20 ng/ml) Lin- cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133+ cells. Therefore, our Lin- stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well-characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.     

Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype

Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation. Robo4 mRNA was specifically expressed in murine HSCs and the immature progenitor cell fraction but not in lineage-positive cells or differentiated progenitors. Moreover, flow cytometry showed a correlation between higher expression of Robo4 and immature phenotypes of hematopoietic cells. Robo4(high) hematopoietic stem/progenitor cells presented higher clonogenic activity or long-term repopulating activity by colony assays or transplantation assays, respectively. A ligand for Robo4, Slit2, is specifically expressed in bone marrow stromal cells, and its expression was induced in osteoblasts in response to myelosuppressive stress. Interestingly, overexpression of Robo4 or Slit2 in HSCs resulted in their decreased residence in the c-Kit(+)Sca-1(+)Lineage(-)-side population fraction. These results indicate that Robo4 is expressed in HSCs, and Robo4/Slit2 signaling may play a role in HSC homeostasis in the bone marrow niche.     

Vascular endothelial cells produce soluble factors that mediate the recovery of human hematopoietic stem cells after radiation injury.

The risk of terrorism with nuclear or radiologic weapons is considered to be high over the coming decade. Ionizing radiation can cause a spectrum of hematologic toxicities, from mild myelosuppression to myeloablation and death. However, the potential regenerative capacity of human hematopoietic stem cells (HSCs) after radiation injury has not been well characterized. In this study, we sought to characterize the effects of ionizing radiation on human HSCs and to determine whether signals from vascular endothelial cells could promote the repair of irradiated HSCs. Exposure of human bone marrow CD34+ cells to 400 cGy caused a precipitous decline in hematopoietic progenitor cell content and primitive cells capable of repopulating nonobese diabetic/severe combined immunodeficient mice (SCID-repopulating cells), which was not retrievable via treatment with cytokines. Conversely, culture of 400 cGy-irradiated bone marrow CD34+ cells with endothelial cells under noncontact conditions supported the differential recovery of both viable progenitor cells and primitive SCID-repopulating cells. These data illustrate that vascular endothelial cells produce soluble factors that promote the repair and functional recovery of HSCs after radiation injury and suggest that novel factors with radiotherapeutic potential can be identified within this milieu.     

Antibodies to stem cell marker antigens reduce engraftment of hematopoietic stem cells

Hematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca-1(+)c-Kit(+)Lin(-) (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell-associated genes c-Kit, Cd34, Tal-1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long-term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody-based stem cell isolation procedures can have negative effects on HSC physiology.     

体外定向诱导小鼠胚胎干细胞发育为造血干/祖细胞方法的初步研究

目的:探讨体外定向诱导胚胎干细胞(ESC)发育为造血干细胞(HSC)的方法。方法:将小鼠E14胚胎干细胞在含干细胞生长因子(SCF)和血管内皮生长因子(VEGF)的甲基纤维素培养基中首先诱导发育为胚胎体(EB),再将EB置于均含SCF、VEGF、IL-3、IL-6及促红细胞生成素(EPO)的3种不同培养体系中定向分化为HSC,并观察HSC表面标志性抗原、造血集落形成及瑞氏-姬姆萨染色的结果。结果:经两阶段诱导ESC分化为HSC,发现在甲基纤维素半固体培养体系中HSC发育缓慢,分化14d后CD34+/Sca-1+细胞数最高为(31.5±4.7)%;而在骨髓基质细胞饲养层上HSC发育较快,细胞数量较多,分化第10dCD34+/Sca-1+细胞数即达到峰值,为(47.8±6.3)%;骨髓基质细胞饲养层+胎肝基质细胞上清培养体系中HSC发育同样迅速,所产生的CD34+/Sca-1+细胞数量在3个体系中最高,为(53.6±7.2)%。经瑞氏-姬姆萨染色证实上述细胞为早期造血细胞,均有形成各系造血细胞集落的能力。结论:使用骨髓基质细胞饲养层+胎肝基质细胞上清培养体系及SCF、VEGF、IL-3、IL-6及EPO等细胞因子,通过两阶段诱导分化,可从小鼠ESC获得较高比例的HSC。     

含人AGM区、胎肝及骨髓基质细胞培养体系程序化诱导小鼠胚胎干细胞向造血干细胞的分化

背景：前期已分别制备人主动脉-性腺-中肾区基质细胞系及胎肝基质细胞系,发现前者可促进小鼠胚胎干细胞定向分化为造血干细胞。 目的：模拟胚胎发育过程中永久造血发育的时空顺序,探讨人主动脉-性腺-中肾(AGM)区、胎肝(FL)及骨髓(BM)基质细胞对小鼠胚胎干细胞体外诱导分化为造血干细胞的支持作用,以寻求更佳的诱导条件。 方法：将小鼠E14胚胎干细胞诱导为拟胚体(EB),并利用Transwell非接触共培养体系依次在人主动脉-性腺-中肾区、胎肝及骨髓基质细胞饲养层上进一步诱导分化,按不同诱导阶段分为拟胚体对照、EB/AGM、EB/AGM+FL和EB/AGM+FL+BM共4组。共培养6 d后分别收获各组拟胚体来源细胞,以流式细胞仪检测Sca-1+c-Kit+细胞含量,进行各系造血细胞集落形成单位分析并观察细胞形态。 结果与结论：①EB/AGM+FL组和EB/AGM+FL+BM组收获细胞涂片均发现原始造血细胞。②拟胚体来源细胞经AGM区基质细胞诱导后Sca-1+c-Kit+ 细胞明显升高(P < 0.05)。③拟胚体对照组造血细胞集落形成单位低于其他各组(P < 0.05), 而EB/AGM+FL、EB/AGM+FL+BM组造血细胞集落形成单位计数亦较EB/AGM组明显增高。提示AGM+FL和AGM+FL+骨髓基质细胞微环境对原始造血干细胞的扩增效应均明显高于单纯主动脉-性腺-中肾饲养层。     

Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells

To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c-kit+ Thy1.1lo Lin- Sca-1+ hematopoietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker-expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor-derived myofibers expressed both donor-specific and host-specific markers, suggesting that they arise by low-level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage.     

Transplantation of bone marrow-derived very small embryonic-like stem cells attenuates left ventricular dysfunction and remodeling after myocardial infarction

Adult bone marrow (BM) contains Sca-1+/Lin-/CD45- very small embryonic-like stem cells (VSELs) that express markers of several lineages, including cardiac markers, and differentiate into cardiomyocytes in vitro. We examined whether BM-derived VSELs promote myocardial repair after a reperfused myocardial infarction (MI). Mice underwent a 30-minute coronary occlusion followed by reperfusion and received intramyocardial injection of vehicle (n= 11), 1 x 10(5) Sca-1+/Lin-/CD45+ enhanced green fluorescent protein (EGFP)-labeled hematopoietic stem cells (n= 13 [cell control group]), or 1 x 10(4) Sca-1+/Lin-/CD45- EGFP-labeled cells (n= 14 [VSEL-treated group]) at 48 hours after MI. At 35 days after MI, VSEL-treated mice exhibited improved global and regional left ventricular (LV) systolic function (echocardiography) and attenuated myocyte hypertrophy in surviving tissue (histology and echocardiography) compared with vehicle-treated controls. In contrast, transplantation of Sca-1+/Lin-/CD45+ cells failed to confer any functional or structural benefits. Scattered EGFP+ myocytes and capillaries were present in the infarct region in VSEL-treated mice, but their numbers were very small. These results indicate that transplantation of a relatively small number of CD45- VSELs is sufficient to improve LV function and alleviate myocyte hypertrophy after MI, supporting the potential therapeutic utility of these cells for cardiac repair. Disclosure of potential conflicts of interest is found at the end of this article.