In-vitro Behavior of Human Umbilical Cord Blood Stem Cells Towards Serum Based Minimal Cytokine Growth Conditions

Abstract

We tried here to optimize the proliferation of both Hematopoietic and Mesenchymal stem cells of Umbilical Cord blood in minimal cytokine growth condition. Failing to get good results of expansion of non-adherent Hematopoietic Total Nucleated Cells and adherent Fibroblastic Mesenchymal Stem Cells derived from 10-12 ml of collected Cord blood, we designed the further experimental study by increasing the volume of Cord blood sample up to 65-70 ml. We harvested the non-adherent as well as adherent fraction separately derived from the primary culture of Umbilical Cord blood stem cells under the influence of growth promoting Cytokines or Growth Factors. The proliferation study was conducted by taking different combinations of two hematopoietic growth stimulatory Cytokines like stem cell factor (SCF) and Fms like tyrosine kinase-3Ligand (Flt3L) at concentrations (10 ng/ml, 100 ng/ml) while we preferred Mesenchymal specific growth factor i.e. basic Fibroblast growth factor (FGF-β) at its 10 ng/ml concentration for adherent cells to get optimal results. The Hematopoietic and Fibroblast Colony forming abilities of the expanded stem cells were performed through Colony Forming Unit assay. Culture Medium containing cytokine combination like SCF 100 ng/ml with Flt3L 10 ng/ml was found to be optimal for the proliferation of hematopoietic stem cells. But the number of hematopoietic colonies like Erythroid colonies generated were less in case of media supplemented with SCF & Flt3L while more number of Myeloid colonies were observed in Growth factor supplemented media in comparison to the control one. The FGF-β supplemented media successfully enhanced the proliferation of Mesenchymal Stem Cells and exhibited its efficient Fibroblast colony forming ability. Our experimental study supports the minimal utilization of cytokines for haematopoietic and mesenchymal stem cell proliferation which may help in future safe Cord blood stem cell infusion.

Keywords: Adherent cells; Cytokines; Hematopoietic stem cells (HSCs); Mesenchymal stem cells (MSCs); Non-adherent cells.

Fig. 1

PrimaryCulture of MNCs derived from 12 ml of Cord blood. a Phase contrast Microscopy image of macrophages &fibroblasts in adherent fraction. b Culture of nonadherent cells c FACS analysis of nonadherent cells. d Culture of adherent cells e FACS analysis of adherent cells

Fig. 2

Higher frequency of fibroblast cells as observed in 65–70 ml of cord blood

Fig. 3

a Confluence attained by proliferated TNCs under the influence of SCF & Flt3L. b Wreight Giemsa Staining. c Comparative analysis of expansion of TNCs between control and GFs stimulated media

Fig. 4

Images of proliferated MSCs in FGF-β supplemented media in P1 (a) and P5 (b) passages. c Graphical presentation of cell proliferation at each passage

Fig. 5

a BFU-E. b CFU-GM. c CFU-GEMM. d Graphical Presentation of mean number of hematopoietic colonies in percentage. e Fibroblast colonies in FGF supplemented media