The Proliferation and Stemness of Peripheral Blood-Derived Mesenchymal Stromal Cells Were Enhanced by Hypoxia

Abstract

This study aimed to address the dilemma of low peripheral blood-derived mesenchymal stromal cell (PBMSC) activity and reduced phenotype in bone or cartilage tissue engineering. Rat PBMSCs (rPBMSCs) were obtained by density gradient centrifugation, and stromal cell characteristics were confirmed by flow cytometry (FCM) and multi-differentiation potential induction experiments. Cell growth curve, viability experiments, and clone formation experiments were performed by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) and cell counting, and the cell cycle was confirmed by cell FCM. The proliferation signal pathway and stemness-related proteins were detected by molecular methods including Western blot and real-time polymerase chain reaction. CD73, CD90, and CD105 were highly expressed, and CD14, CD19, CD34, CD45, and HLA-DR were barely expressed in rPBMSCs. rPBMSCs possessed the potential to differentiate into chondrocytes, adipocytes, and osteoblasts under their respective induction conditions. Cell growth curve and viability experiments were performed under hypoxic conditions: 19% O₂, 5% O₂, and 1% O₂. Specifically, 5% O₂ accelerated the proliferation and expression of the stemness of PBMSCs. Cycle experiments proved that hypoxia promoted the cell transition from the G1 phase to the S phase. Molecular experiments confirmed that 5% O₂ hypoxia significantly elevated the expressions of hypoxia-inducible factor 1α and β-catenin and simultaneously the expressions of cycle-related genes including CyclinE/CDK2 and stemness-related genes including Nanog and SOX2. The appropriate concentration of hypoxia (i.e., 5% O₂) enhanced the proliferation and stemness of rPBMSCs and increased the multidirectional differentiation potential of stromal cells. The proposed culture method could improve the viability and maintain the phenotype of rPBMSCs in cartilage or bone tissue engineering.

Keywords: HIF-1α; hypoxia; peripheral blood-derived mesenchymal stromal cells (PBMSCs); proliferation; stemness.

Figure 1

Characteristics of rPBMSCs. (A) Morphology of rPBMSCs cultured for 1 day, 7 days, and 21 days. (B) Immunocytochemical staining demonstrated a positive expression of CD73, CD90, and CD105 and negative expressions of CD14, CD19, CD34, CD45 CD45, and HLA-DR. (C) Multilineage differentiation capacities of rPBMSCs. Magnification, 200×. rPBMSCs, rat peripheral blood-derived mesenchymal stromal cells.

Figure 2

Effect of hypoxia on rPBMSC proliferation. (A) The cell growth curves of third-generation PBMSCs (P3 PBMSCs), fifth-generation PBMSCs (P5 PBMSCs), and sixth-generation PBMSCs (P6 PBMSCs) were drawn based on the number of cells counted at each time point, the cumulative population doubling curve of sixth-generation PBMSCs (P6 PBMSCs) was determined based on cell culture time. (B) Absorbance of P3, P5, and P6 rPBMSCs treated under normoxia and hypoxia (5% O₂) at Day 8. (C) Hypoxia increased the number of rPBMSC colonies. (D) Measurement of the number of colonies in each group. All data are presented as means ± SEM. P < 0. 05; ^∗ vs control group. rPBMSCs, rat peripheral blood-derived mesenchymal stromal cells.

Figure 3

Hypoxia promoted cell cycle transition and maintained the trilineage differentiation capacity of rPBMSCs. (A, B) Hypoxia promoted cell cycle transition, as determined by flow cytometry. (C) rPBMSCs were cultured under normoxia and hypoxia (5% O₂) for chondrogenic differentiation, osteogenic differentiation, and adipogenic differentiation for 21 days. Magnification, 200×. Arrows indicate lipid droplets, proteoglycans, and calcium nodules. rPBMSCs, rat peripheral blood-derived mesenchymal stromal cells.

Figure 4

Immunoassay of HIF-1α and β-catenin in rPBMSCs under normoxia and hypoxia (5% O₂). (A) Nuclear expression of HIF-1α (red) and β-catenin (green) in rPBMSCs treated under normoxia and hypoxia (5% O₂) for 5 days. (B) Nuclear expression of HIF-1α (brown) and β-catenin (brown) in rPBMSCs treated under normoxia and hypoxia (5% O₂) for 5 days. (C, D) Quantitative analysis of HIF-1α (brown) and β-catenin (brown) in rPBMSCs in panel (B). Magnification, 200×. All data are presented as means ± SEM. P < 0. 05; ^∗ vs control group. rPBMSCs, rat peripheral blood-derived mesenchymal stromal cells; HIF-1α, hypoxia-inducible factor 1α.

Figure 5

Hypoxia regulates the expressions of cycle-related and self-renewal-related molecules. (A–C) mRNA and protein expression levels of β-catenin, Cyclin E, and CDK2 in rPBMSCs treated under normoxic and hypoxic (5% O₂) conditions for 5 days. (D–F) mRNA and protein expression levels of HIF-1α, Nanog, and SOX2 in rPBMSCs treated under normoxic and hypoxic (5% O₂) conditions for 24 h. All data are presented as means ± SEM. P < 0. 05; ^∗ vs control group. rPBMSCs, rat peripheral blood-derived mesenchymal stromal cells; HIF-1α, hypoxia-inducible factor 1α.

Figure 6

Mechanism of hypoxia-promoted proliferation and stemness of rPBMSCs.