Rationale:: Bone marrow (BM)–derived mesenchymal stem cells (MSCs) hold great promise for cardiovascular cell therapy owing to their multipotency and culture expandability. Objective:: The aim of the study was to investigate whether MSCs can treat experimental acute myocardial infarction (MI) and diabetic neuropathy. Methods and Results:: We isolated mononuclear cells from mouse BM and cultured MSCs in a conventional manner. Flow cytometry analyses of these cultured cells at passage 4 showed expression of typical MSC markers such as CD44 and CD29, but not hematopoietic markers such as c-kit, flk1, and CD34. To determine the therapeutic effects of MSCs, we injected MSCs into the peri-infarct area after ligation of the left anterior descending coronary arteries of mice and, as separate experiments, injected the same batch of MSCs into hindlimb muscles of mice with diabetic neuropathy. During the follow-up at 4 to 8 weeks after cell transplantation, growing tumors were observed in 30% of hearts in the MI model, and in 46% of hindlimbs in the diabetic neuropathy model. Histological examination of the tumors revealed hypercelluarity, pleomorphic nucleoli, cytological atypia and necrosis, and positive staining for α-smooth muscle actin, indicative of malignant sarcoma with myogenic differentiation. Chromosomal analysis of these MSCs showed multiple chromosomal aberrations including fusion, fragmentation, and ring formation. Conclusions:: Genetically unmodified MSCs can undergo chromosomal abnormalities even at early passages and form malignant tumors when transplanted in vivo. These results suggest that careful monitoring of chromosomal status is warranted when in vitro expanded MSCs are used for cell therapy such as for MI.

Rationale:: Bone marrow (BM) cells play an important role in physiological and therapeutic neovascularization. However, it remains unclear whether any specific uncultured BM cell populations have higher angiogenic and vasculogenic activities. Moreover, there has been controversy regarding the vasculogenic ability of BM cells. Objective:: Preliminary flow cytometric analysis showed that CD31, traditionally a marker for endothelial cells, is expressed in certain nonendothelial BM mononuclear cells in both human and mouse. Based on the conserved CD31 expression in the axis of hematopoietic stem/progenitor cells (HSC/HPCs) to endothelial cells, we further sought to determine the comprehensive vasculogenic and angiogenic characteristics of human and mouse BM-derived CD31 ⁺ cells. Methods and Results:: Flow cytometric analysis demonstrated that all CD31 ⁺ cells derived from BM were CD45 ⁺ and expressed markers for both HSC/HPCs and endothelial cells. Comprehensive gene expression analyses revealed that BM-CD31 ⁺ cells expressed higher levels of angiogenic genes than CD31 ⁻ cells. Endothelial progenitor cells, as well as HSC/HPCs, were almost exclusively confined to the CD31 ⁺ cell fraction, and culture of CD31 ⁺ cells under defined conditions gave rise to endothelial cells. Finally, injection of CD31 ⁺ cells into ischemic hindlimb repaired ischemia, increased expression of angiogenic and chemoattractive factors, and, in part, directly contributed to vasculogenesis, as demonstrated by both 3D confocal microscopy and flow cytometry. Conclusions:: These data indicate that BM-CD31 ⁺ cells represent highly angiogenic and vasculogenic cells and can be a novel and highly promising source of cells for cell therapy to treat ischemic cardiovascular diseases.

Rationale:: Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. Objective:: We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. Methods and Results:: We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR ⁺ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks’ transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. Conclusions:: This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.

Background—: Emerging evidence has suggested a contribution of bone marrow (BM) cells to lymphatic vessel formation; however, the exact phenotype of the cells with lymphatic endothelial progenitor cell function has yet to be identified. Here, we investigate the identity of BM-derived lymphatic endothelial progenitor cells and their role in lymphatic neovascularization. Methods and Results—: Culture of BM-mononuclear cells in the presence of vascular endothelial growth factors A and C and endothelial growth factor resulted in expression of lymphatic endothelial cell markers. Among these cells, podoplanin ⁺ cells were isolated by magnetic-activated cell sorting and characterized by fluorescence-activated cell sorter analysis and immunocytochemistry. These podoplanin ⁺ cells highly express markers for lymphatic endothelial cells, hematopoietic lineages, and stem/progenitor cells; on further cultivation, they generate lymphatic endothelial cells. We further confirmed that podoplanin ⁺ cells exist in small numbers in BM and peripheral blood of normal mice but are significantly (15-fold) augmented on lymphangiogenic stimuli such as tumor implantation. Next, to evaluate the potential of podoplanin ⁺ cells for the formation of new lymphatic vessels in vivo, we injected culture-isolated or freshly isolated BM-derived podoplanin ⁺ cells into wound and tumor models. Immunohistochemistry demonstrated that the injected cells were incorporated into the lymphatic vasculature, displayed lymphatic endothelial cell phenotypes, and increased lymphatic vascular density in tissues, suggesting lymphvasculogenesis. Podoplanin ⁺ cells also expressed high levels of lymphangiogenic cytokines and increased proliferation of lymphatic endothelial cells during coculture, suggesting a lymphangiogenic or paracrine role. Conclusions—: Our results provide compelling evidence that BM-derived podoplanin ⁺ cells, a previously unrecognized cell type, function as lymphatic endothelial progenitor cells and participate in postnatal lymphatic neovascularization through both lymphvasculogenesis and lymphangiogenesis.

Background— Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. Methods and Results— We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow–derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. Conclusions— We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.