Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential.

BACKGROUND: Cell-based therapies involving mononuclear cells (MNCs) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNCs has not been evaluated. We investigated the therapeutic potential of peripheral blood (PB) MNCs, operated by recently developed quality and quantity (QQ) culture of endothelial progenitor cells (EPCs). METHODS AND RESULTS: PBs were collected from healthy volunteers; peripheral blood mononuclear cells (PBMNCs) isolated from these PBs were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6. The resulting cells (QQMNCs) in EPC colony-forming assay generated significantly more definitive EPC colonies than PBMNCs. In flow cytometry, macrophages and helper T lymphocytes of QQMNCs became phenotypically polarized into angiogenic, anti-inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T-cell expansion. Quantitative real-time polymerase chain reaction (qRT-PCR) assay revealed the predominant proangiogenic gene expressions in QQMNCs versus PBMNCs. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured "early EPC" Tx (eEPCTx), and granulocyte colony-stimulating factor mobilized CD34(+) cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx. Histological evaluations and qRT-PCR assays in ischemic hindlimbs demonstrated that QQMNCTx, similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx. CONCLUSIONS: QQ culture potentiates the ability of PBMNCs to promote regeneration of injured tissue; considering the feasible cell preparation, QQ culture-treated PBMNCs may provide a promising therapeutic option for ischemic diseases. CLINICAL TRIAL REGISTRATION URL: irb.med.u-tokai.ac.jp/d/2/monthly/2010.html; IRB No.: 10R-020.URL: irb.med.u-tokai.ac.jp/d/2/monthly/201312.html; IRB No.: 13R228.

Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment.

Hematopoiesis is maintained by specific interactions between both hematopoietic and nonhematopoietic cells. Whereas hematopoietic stem cells (HSCs) have been extensively studied both in vitro and in vivo, little is known about the in vivo characteristics of stem cells of the nonhematopoietic component, known as mesenchymal stem cells (MSCs). Here we have visualized and characterized human MSCs in vivo following intramedullary transplantation of enhanced green fluorescent protein-marked human MSCs (eGFP-MSCs) into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Between 4 to 10 weeks after transplantation, eGFP-MSCs that engrafted in murine BM integrated into the hematopoietic microenvironment (HME) of the host mouse. They differentiated into pericytes, myofibroblasts, BM stromal cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells, which constituted the functional components of the BM HME. The presence of human MSCs in murine BM resulted in an increase in functionally and phenotypically primitive human hematopoietic cells. Human MSC-derived cells that reconstituted the HME appeared to contribute to the maintenance of human hematopoiesis by actively interacting with primitive human hematopoietic cells.