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

Weiping Lin; Liangliang Xu; Sien Lin; Liu Shi; Bin Wang; Qi Pan; Wayne Y W Lee; Gang Li

doi:10.1016/j.jot.2019.02.003

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

J Orthop Translat. 2019 Mar 7:19:18-28. doi: 10.1016/j.jot.2019.02.003. eCollection 2019 Oct.

Authors

Weiping Lin^{1

2}, Liangliang Xu³, Sien Lin^{1

2}, Liu Shi^{1

2}, Bin Wang^{1

2}, Qi Pan^{1

2}, Wayne Y W Lee^{1

2}, Gang Li^{1

2

4}

Affiliations

¹ Department of Orthopaedics & Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, PR China.
² Stem Cells and Regenerative Medicine Laboratory, Lui Che Woo Institute of Innovative Medicine, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, PR China.
³ Key Laboratory of Orthopaedics & Traumatology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, The First Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, PR China.
⁴ The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, PR China.

Abstract

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 β-tricalcium phosphate (β-TCP) may be used to promote bone repair.

Keywords: Bone regeneration; Human peripheral blood; Hypoxia; Lgr5; Mesenchymal stem cells; Multidifferentiation; Protocol.