BackgroundA culture system that closely recapitulates marrow physiology is essential to study the nichemediated regulation of hematopoietic stem cell fate at a molecular level. We investigated the key features that play a crucial role in the formation of a functional niche in vitro. Design and MethodsHydrogel-based cultures of human placenta-derived mesenchymal stromal cells were established to recapitulate the fibrous three-dimensional architecture of the marrow. Plastic-adherent mesenchymal stromal cells were used as controls. Human bone marrow-derived CD34 + cells were co-cultured with them. The output hematopoietic cells were characterized by various stem cell-specific phenotypic and functional parameters. ResultsThe hydrogel-cultures harbored a large pool of primitive hematopoietic stem cells with superior phenotypic and functional attributes. Most importantly, like the situation in vivo, a significant fraction of these cells remained quiescent in the face of a robust multi-lineage hematopoiesis. The retention of a high percentage of primitive stem cells by the hydrogel-cultures was attributed to the presence of CXCR4-SDF1α axis and integrin beta1-mediated adhesive interactions. The hydrogel-grown mesenchymal stromal cells expressed high levels of several molecules that are known to support the maintenance of hematopoietic stem cells. Yet another physiologically relevant property exhibited by the hydrogel cultures was the formation of hypoxia-gradient. Destruction of hypoxia-gradient by incubating these cultures in a hypoxia chamber destroyed their specialized niche properties. ConclusionsOur data show that hydrogel-based cultures of mesenchymal stromal cells form a functional in vitro niche by mimicking key features of marrow physiology.

Critical limb ischemia (CLI) is the end stage of lower extremity peripheral vascular disease (PVD) in which severe obstruction of blood flow results in ischemic rest pain, ulcers and/or gangrene, and a significant risk of limb loss. This open-label, single-arm feasibility study evaluated the safety and therapeutic effectiveness of autologous bone marrow cell (aBMC) concentrate in revascularization of CLI patients utilizing a rapid point-of-care device. Seventeen (17) no-option CLI patients with ischemic rest pain were enrolled in the study. Single dose of aBMC, prepared utilizing an intraoperative point-of-care device, the Res-Q™ 60 BMC system, was injected intramuscularly into the afflicted limb and patients were followed up at regular intervals for 12 months. A statistically significant improvement in Ankle Brachial Index (ABI), Transcutaneous Oxygen Pressure (TcPO2), mean rest pain and intermittent claudication pain scores, wound/ ulcer healing, and 6-minute walking distance was observed following aBMC treatment. Major amputation-free survival (mAFS) rate and amputation-free rates (AFR) at 12 months were 70.6% and 82.3%, respectively. In conclusion, aBMC injections were well tolerated with improved tissue perfusion, confirming the safety, feasibility, and preliminary effectiveness of aBMC treatment in CLI patients.

Quiescent cells pose a formidable challenge in clinical management of leukemia, since they escape chemo-radiotherapy and become a source of post-therapy relapse. These cells may be refractory to various known growth-promoting signals, making it imperative to identify the biochemical signals necessary to coax them into mitosis. Using serum-starved cell lines as an experimental model of quiescent leukemic cells (QLCs), we demonstrate that a suppression of p38 stress kinase by pharmacological means forms a sufficient trigger to induce proliferative responses in the treated QLCs, even in the absence of any external growth-promoting stimulus. A robust expression of Ki67 and B23 was seen in treated cells, an effect clearly mediated through the activation of extra-cellular signal-regulated kinase (MEK/ERK) pathway. Commensurate with their proliferative status, the treated QLCs got sensitized to significantly low concentrations of anti-mitotic agents. Most importantly, primitive leukemic progenitors present in the mononuclear cells (MNCs) that were isolated from the peripheral blood of freshly diagnosed untreated acute myeloid leukemic (AML) patients got more efficiently killed by cytosine arabinoside (AraC), when the cells were pre-treated with a pharmacological inhibitor of p38. Our data strongly suggest that a suppression of p38 leads to the sensitization of QLCs to anti-mitotic drugs by triggering proliferative responses in them. This approach may have a potential clinical application.