Reprogramming of blood cells into induced pluripotent stem cells as a new cell source for cartilage repair

Abstract

Background: An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair.

Methods: We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression.

Results: hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC-MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC-MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells.

Conclusions: This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.

Fig. 1

Generation of hiPSCs from peripheral blood. a Phase-contrast image of the PBCs. Scale bar = 100 μm. b Phase-contrast image of a hiPSC line. Scale bar = 100 μm. c Karyotype (46,XY). d Fluorescent immunostaining of pluripotent markers OCT4, SSEA4, TRA-1-60, NANOG, and TRA-1-81. Scale bars = 100 μm. e Teratoma formation. Teratomas were harvested over 2 months after subcutaneous injection of the iPSCs into SCID mice. (i) Neuroepithelial cells, (ii) cartilage, and (iii) respiratory epithelium were detected by hematoxylin and eosin staining. Scale bar = 50 μm

Fig. 2

Generation of hiPSC–MSC-chondrogenic pellets via EB formation, monolayer cell culture and three-dimensional pellet culture. a EB formation. Scale bar = 100 μm. b Cell outgrowth from EBs. Scale bar = 100 μm. c Monolayer cell culture. Scale bar = 100 μm. d Three-dimensional pellet culture

Fig. 3

Flow cytometric analysis of the MSC markers (CD73, CD105) and hematopoietic markers (CD34, CD45) in monolayer-cultured hiPSC–MSC-like cells. a Immunological control for CD45, CD73. b Immunological control for CD34, CD105. c, d The cell surface markers CD34 and CD45 were not expressed. e The proportion of CD73-expressing cells was 81.81 ± 2.05 %. f The proportion of CD105-expressing cells was 81.90 ± 1.61 %. Values represent means ± SEM; n = 3. CD cluster of differentiation, Ig immunoglobulin

Fig. 4

Characterization of hiPSC–MSC chondrogenic pellets. a Alcian blue staining and b toluidine blue staining of glycosaminoglycans and proteoglycans revealed the chondrocyte-type appearance of the hiPSC–MSC chondrogenic pellets. Scale bars = 100 μm. c, d Immunohistochemistry for type II and type X collagen. Scale bars = 100 μm. e Biochemical characterization of hiPSC–MSC chondrogenic pellets (hiPSC-Chon) versus hiPSCs, EBs, hiPSC–MSC-like cells (hiPSC–MSCs) and hMSC chondrogenic pellets (hMSC-Chon) versus hMSCs. sGAG per DNA. Bars represent means ± SEM; n = 3; *P < 0.05. EB embryoid body, hiPSC human induced pluripotent stem cell, hMSC human mesenchymal stem cell, sGAG sulfated glycosaminoglycan

Fig. 5

Gene expressions of chondrogenic markers in hiPSC–MSC chondrogenic pellets. The expressions of COL2, COL10, SOX9 and AGGRECAN of hiPSC–MSC chondrogenic pellets (hiPSC-Chon) versus hiPSCs and hiPSC–MSC-like cells (hiPSC–MSCs), compared with hMSC pellets (hMSC-Chon) versus hMSCs. The expression levels of COL2, COL10, SOX9 and AGGRECAN were analyzed by real-time quantitative PCR relative to β-ACTIN. Bars represent means ± SEM; n = 3; *P < 0.05. hiPSC human induced pluripotent stem cell, hMSC human mesenchymal stem cell