An optimization of hematopoietic stem and progenitor cell isolation for scientific and clinical purposes by the application of a new parameter determining the hematopoietic graft efficacy.

Abstract

Abstract:The transplantation of hematopoietic stem and progenitor cells (HSPC) is an established lifesaving therapy. Bone marrow (BM), harvested from heparinized cadaveric organ donors, peripheral blood (PB) and cord blood (CB), are important sources of hematopoietic stem cells. HSPCs, which are used for transplantation purposes, are routinely evaluated in terms of number of mononuclear cells (MNCs), CD34 + MNCs count and viability. The efficacy of grafting is determined additionally in clonogenic tests in vitro. These tests deliver important information about the number of HSPCs and their proliferative potential. Unfortunately, they do not give a possibility to evaluate the functional HSPC chemotactic reactivity in the SDF-1 gradient, which is probably the key phenomenon for HSPC homing after transplantation procedure. Thus, the aim of our study was to optimize HSPC isolation according to their chemotactic reactivity in SDF-1 gradient. Using multiparameter cell sorter (FACS Aria, BD) we examined the HSPCs attracted by SDF-1 on a single cell level. The population of cells which participated in the chemotactic process was highly enriched in CXCR4 + lin -AC133 + CD45 + cells (referred as hematopoietic stem cells) and to our surprise in CXCR4 + lin -AC133 + CD45 -cells (referred as pluripotent stem cells) in quantitative amounts. Since reactivity of HSPCs may depend on various factors involved in the protocol of their isolation and short-term storage, we tested the most commonly used anticoagulants (ACD, CPDA-1, EDTA and Heparin) and culture media (DME, IMDM, RPMI). HSPCs, harvested from CB, PB and BM, were subsequently investigated for clonogenic growth of CFU-GM in methylcellulose cultures and for the level of apoptosis by employing annexin V staining. Evaluating clonogenic potential, ability of chemotactic reactivity in SDF-1 gradient and intensification of apoptosis of HSPC as the most safe anticoagulant and medium were selected. This study has proved that chemotactic reactivity of HSPCs is a new but very important parameter which should be included in the procedure of their isolation.

The Ins and Outs of Hematopoietic Stem Cells: Studies to Improve Transplantation Outcomes

“…Although number of HSPC plays a large part in determining the ultimate function of the graft, HSPC quality is likewise important for engraftment results. Graft characteristics, defined as the cell types found in the graft, their gene expression profiles, and their chemotactic reactivity to an SDF-1 gradient, all influence BM homing, engraftment, immune reconstitution and other post-transplant outcomes [153, 154]. …”

Anticoagulants on yield of bone marrow-derived mononuclear cells harvested from dogs

“…In study focusing on the influence of different anticoagulants on chemotactic reactivity, the heparin has been considered the most suitable and safe anticoagulant for freshly isolated bone marrow hematopoietic stem and progenitor cells (BaUMERT et al, 2008). In this study, we have not found significant differences at harvest in terms of stem cell yield with both anticoagulants tested.…”

“…heparin and CPDa-1 are commonly used to harvest bone marrow (BaUMERT et al, 2008). heparin is a potent anticoagulant that has effects only in the presence of a plasma component termed heparincofactor (ROSENBERG, 1974), while the CPDa-1 is an adenine-containing preservative solution used to anticoagulant blood over a wider range (BEUTlER & WEST, 1979).…”

The Effect of Intracoronary Infusion of Autologous Bone Marrow-Derived Lineage-Negative Stem/Progenitor Cells on Remodeling of Post-Infarcted Heart in Patient with Acute Myocardial Infarction

“…BM aspirates were obtained from 15 patients in study group under local anesthesia from the posterior iliac crest within 24 hours after PCI. Mononuclear cells (MNCs) were obtained from harvested BM using centrifugation over Gradisol L (Polfa, Kutno, Poland) as described elsewhere [21]. Subsequently, the cells were incubated with anti-human antibodies conjugated with microbeads using a Lineage Depletion Kit (Myltenyi Biotec, Auburn, AL, USA) and isolated according to manufacturer's protocol as described [17], according to the good medical practice conditions.…”