Abstract

 Background and Objectives

 The isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures are arduous due to the unwanted growth of hematopoietic cells and non-MSCs. In this study, homogenous populations of CD34⁺ MSCs from mouse bone marrow were purified through positive immunoselection.

 Materials and Methods

 In this experimental study, C57BL/6 mice were sacrificed. Their bone marrow cells were aspirated and incubated with anti-CD34 conjugated magnetic beads. After immunoselection, a sample of the cells was prepared for flow cytometry in order to examine the expression of CD34 antigen and the remains were cultivated. 24 hours later, non-adherent cells, mostly consisting of CD34⁺ hematopoietic stem cells were removed and the plastic adherent cultivated cells were investigated for surface markers (CD44, Sca-1, Vcam-1, CD34, CD11b and CD45). The plastic adherent cultivated cells were differentiated into the osteoblastic and adipogenic lineages to investigate the mesenchymal nature. Furthermore, the expression of some surface markers were investigated through flow cytometry.

 Results

 Purified populations of fibroblast-like CD34⁺ cells were achieved in the first passage (one week after initiative cultivation). The CD34, CD44, Sca-1 and Vcam-1 markers were expressed but CD11b and CD45 were absent. Osteocyte (osteocalsin, osteopontin, parathyroid hormone receptor) and adipocyte (lipo protein lipase and adipsin) differentiating genes were expressed in these cells.

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 Conclusions

 This study indicates that this protocol can result in efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We showed that plastic adherent murine bone marrow derived CD34⁺ cells with capability of differentiating into skeletal lineages in vitro are MSCs.

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 Key words: Mesenchymal stem cells, Bone marrow, CD34 antigen

  Abstract

 Background and Objectives

 Stem cell transplantation has achieved high success for treatment but the use of this source has some limitation. Cord blood transplantation is clinically limited by its low progenitor cell contents such as CD34⁺. The aim of this study was to evaluate the effect of mesenchymal stem cells (MSCs) on replication of CD34⁺ in cord blood.

 Materials and Methods

 In this experimental study MSCs were isolated and cultured from BM mononuclear cells. MSCs were used for co-culture in two systems of 2D and 3D cultures. After isolation, umbilical cord blood CD34⁺ cells were cultured in two different systems. The level of expansion in these systems was evaluated by cell count, analysis of CD34⁺ expression by flowcytometery, and CFU assay .

 Results

 Expansion of cord blood HSCs was evaluated at three different culture systems at three different times (day3, day 7 and day 10): the medium with cytokine, 2D medium with cytokine + MSCs, and 3D medium with cytokine + MSCs on scaffold. The fold increase of TNC, CFU, and CD34⁺ cells at day 10 for the former medium was 87 ± 6, 84 ± 8, 11 ± 3, for the second 103 ± 8, 111 ± 9, 14 ± 3, and for the latter 127 ± 10, 170 ± 14, 20 ± 4, respectively. The highest expansion was observed in the 3D co-culture with MSCs after 10 days.

 Conclusions

 The results have demonstrated that the co-culture of CB HSCs with mesenchymal cells could increase the rate of expansion especially in the 3D system. 

  Abstract

 Background and Objectives

 Umbilical cord blood is a promising alternative source of hematopoietic stem/progenitor cells (HSPCs) for allogeneic hematopoietic stem cell transplantation. Although the use of UCB for transplantation has plentiful advantages in comparison with other cell sources, the low number of HSPCs led to cell delayed recovery. Therefore, ex vivo expansion of HSPC before transfusion could induce faster cell recovery.

 Materials and Methods

 CD34 positive cells were positively enriched using MidiMACS system and cultured on aminated PS nanofibers, aligned PCLs, and random PCLs. Cell expansion and quantification of cell apoptosis were carried out in expanded cell populations in different conditions by the flowcytometric method.

 Results

 This study indicated significantly increased expansion of CD34⁺ cells and declined apopotosis in PS-cultured cells compared to the same cells on 2-D control culture system (p< 0.05). There were no significantly differences in expansion of CD34⁺ cells on aligned and random PCL scaffolds than conventional system.

 Conclusions

 This study demonstrated that aminated PS nanofibers has positive effects on the expansion of CD34⁺ cells compared to 2D culture system by increasing cell expansion and cell apopotosis inhibition. However, aligned and random PCL nanofibers did not show supportive effects on cell proliferation.