Characterisation of multipotent stem cells from human peripheral blood using an improved protocol

Background: A promising approach of bone repair is to use stem cells, such as mesenchymal stem cells (MSCs). Seeking available source of MSCs still remains a great challenge in tissue engineering and cell therapy. Peripheral blood (PB) emerges as an alternative source of MSCs which can be easily acquired with minimal invasiveness. This study was undertaken to evaluate the multipotency of PB-MSCs and effects of human PB-MSCs transplantation on ectopic bone regeneration in nude mice. Methods: Human venous blood collected was mixed with heparin and then red blood cells were removed using red blood cell lysis buffer. Cell suspension was cultured in normoxia-culture and hypoxia-culture conditions, respectively. The non-adherent cells were removed by half changing culture media every three days. Cells were selected due to plastic adherence. The adherent cells were then passaged and subjected to multi-differentiation induction assays in vitro and in vivo ectopic bone formation assay. Results: Characterization assays indicated that cells cultured under hypoxia possessed potent multi-lineage differentiation capacity and expressed Nanog and Lgr5, as well as a series of MSC surface antigens (including CD29, CD90, CD105, and CD73). Additionally, regenerated bone tissues by transplantation of human PB-MSCs in vivo were confirmed by histological examinations of ectopic osteogenesis assay. A purified population of MSCs can be obtained within a short period of time using this protocol with a successful rate of 60%. Conclusion: We reported an effective and reliable method to harvest highly purified MSCs with potent multi-differentiation potential from human peripheral blood. Lgr5 may be a potential biomarker for identification of a subpopulation of PB-MSCs. The translational potential of this article: PB-MSCs is an alternative cell source for cell therapy, which may be harvested, culture expanded and PB-MSCs loaded with 3-tricalcium phosphate (beta-TCP) may be used to promote bone repair. (C) 2019 Chinese University of Hong Kong. Published by Elsevier (Singapore) Pte Ltd on behalf of Chinese Speaking Orthopaedic Society.