6F). (DAPI) in blue.(TIF) pone.0045603.s002.tif (417K) GUID:?0402B058-C3C4-4ACD-9841-4F9072859522 Physique S3: Mouse iPSC-derived CPCs do not illicit an immune response growth that maintain their multipotency. Methodology/Principal Findings We sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1+/Nkx2.5+ CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1+/Nkx2.5+ CPCs and protocols for their clonal growth and transplantation and strong ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs post transplantation into adult hearts. Introduction Despite therapeutic advancements, cardiovascular disease remains a major cause of morbidity and Pulegone mortality worldwide. Although current therapies slow the progression of cardiovascular disease, there are few if any options to reverse or repair Pulegone damaged myocardium. Unfortunately, adult cardiac myocytes (CMs) lack the ability to divide and replace those that are damaged after injury Pulegone in any clinically significant manner [1]. Investigators have been exploring the feasibility of directly injecting stem cells into the heart for therapeutic cell transplantation and regeneration. While multiple animal studies have exhibited the ability of adult stem cells to improve left ventricular function, long-lasting effects, CM differentiation or even engraftment of injected cells has been more difficult to establish [2], [3]. Likewise, early human clinical trials testing the efficacy of adult stem cell therapy to restore perfusion and mechanical function to the heart after myocardial infarction (MI), although promising, have had variable results [4]. Since most preclinical studies have demonstrated very low rates of cardiac differentiation when using these cells [5], there is increasing consensus that transplanted adult stem cells may have a limited capacity for true cardiac regeneration and their beneficial effects are more likely related to paracrine mechanisms [6]. This highlights the need for cell types that can provide long-lasting engraftment and myogenesis either alone or in combination with existing cell types. Embryonic stem cells (ESCs) are a reliable source of authentic CMs, but issues of immunogenicity, oncogenic risk and ethical concerns have hampered their clinical translation. Recent advances in stem cell biology to induce pluripotency in somatic cells make the potential of autologous, regenerative strategies a viable possibility [7]. However, translating the promise of iPSCs into a viable therapy will require the identification and characterization of appropriate iPSC-derived progenitor cells. We believe that the optimal cell type would be lineage-committed, multipotent CPCs that satisfy the need for multilineage differentiation while limiting the oncogenic risk of injecting undifferentiated iPSCs or ESCs. Recently, a multipotent CPC was identified based on the expression of transcription factors Isl1+ and Nkx2.5+ [8], [9] in ESCs and fetal hearts; however, surface markers to identify and enrich for these Isl1+/Nkx2.5+ CPCs are neither specific nor uniformly agreed upon. Previously described cell surface proteins Flk1 and Kit oncogene (c-kit), which have been used in combination to identify mouse CPCs, are not specific markers for endogenous CPCs [10] since Flk1 is usually broadly expressed developmentally on all cardiovascular cell types and not limited to Isl1+/Nkx2.5+ CPCs [11]. Genetically modifying CPCs with integrating viruses to express fluorescent markers under the control of Isl1 or Nkx2. 5 promoters has also been used to identify these CPCs [12]. However, this would complicate their use clinically in human trials due to potential oncogenic risk incurred by genomic manipulation. Therefore, the ability to utilize CPCs derived from human iPSCs therapeutically will require the identification of surface markers to isolate and enrich for Isl1+/Nkx2.5+ CPCs without genetic manipulation [10]. Furthermore, it has proven difficult to propagate and expand progenitor cells while simultaneously maintaining their multipotent differentiation potential, hampering attempts to generate sufficient numbers of CPCs to study and/or use in regenerative therapies. Thus, the lack of specific cell surface markers that identify Isl1+/Nkx2.5+ CPCs in an unmodified form and the lack of appropriate conditions to expand them remains one of the major roadblocks facing translational clinical applications of CPCs [10]. In this study, we attempted Rabbit Polyclonal to PRRX1 to identify cell surface markers that are specific to and allow enrichment of Isl1+/Nkx2.5+ CPCs. We identified Flt1 and Flt4 as a novel cell surface marker combination.