Comparison of Four Protocols to Generate Chondrocyte-Like Cells from Human Induced Pluripotent Stem Cells (hiPSCs)

Abstract

Stem cells (SCs) are a promising approach to regenerative medicine, with the potential to treat numerous orthopedic disorders, including osteo-degenerative diseases. The development of human-induced pluripotent stem cells (hiPSCs) has increased the potential of SCs for new treatments. However, current methods of differentiating hiPSCs into chondrocyte-like cells are suboptimal and better methods are needed. The aim of the present study was to assess four different chondrogenic differentiation protocols to identify the most efficient method of generating hiPSC-derived chondrocytes. For this study, hiPSCs were obtained from primary human dermal fibroblasts (PHDFs) and differentiated into chondrocyte-like cells using four different protocols: 1) monolayer culture with defined growth factors (GF); 2) embryoid bodies (EBs) in a chondrogenic medium with TGF-β3 cells; 3) EBs in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) and 4) EBs in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3. The cells obtained through these four protocols were evaluated and compared at the mRNA and protein levels. Although chondrogenic differentiation of hiPSCs was successfully achieved with all of these protocols, the two fastest and most cost-effective methods were the monolayer culture with GFs and the medium conditioned with human chondrocytes. Both of these methods are superior to other available techniques. The main advantage of the conditioned medium is that the technique is relatively simple and inexpensive while the directed method (i.e., monolayer culture with GFs) is faster than any protocol described to date because it is does not require additional steps such as EB formation.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-016-9708-y) contains supplementary material, which is available to authorized users.

Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter

“…Human-induced pluripotent stem cells (hiPSCs) offer a promising source for cartilage tissue engineering and in vitro disease modeling [7] as they have virtually unlimited expansion capacity, can be genetically modified, and can avoid many of the ethical considerations associated with embryonic stem cells [8,9]. Despite reports of several chondrogenic differentiation protocols for pluripotent stem cells [10][11][12][13][14][15], incomplete differentiation and cell heterogeneity remain as the major obstacles for iPSC chondrogenesis [16,17]. This challenge has been addressed in other stem and progenitor cell types by prospectively isolating cells that exhibit chondrogenic lineage commitment using surface marker expression.…”

Mimetic Hierarchical Approaches for Osteochondral Tissue Engineering

“…Suchorska et al compared four methods to generate chondrocyte-like cells from hiPSCs: (1) monolayer culture with addition of defined mesodermal and chondrogenic growth factors (GFs) (DIRECT protocol), (2) EBs differentiated in chondrogenic medium with TGF-β3 cells (TGF-β3 protocol), (3) EBs differentiated in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) (COND protocol) and (4) EBs differentiated in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3 (TGF-β3 + COND protocol). Two fastest and most cost-effective methods were the monolayer culture with GFs (DIRECT) and the medium conditioned with human chondrocytes (COND) [47]. De Peppo et al engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors, and confirmed their phenotype stability in a subcutaneous implantation model [48].…”

Komórki macierzyste i reprogramowanie komórek somatycznych – wybrane zagadnienia

“…w przypadku urazów rdzenia kręgowego, zawału mięśnia sercowego). Do tej pory uzyskano komórki β trzustki syntetyzujące insulinę [40] [42], neurony [43], chondrocyty [44] oraz wiele innych. Komórki IPS, otrzymane z komórek chorego, można modyfikować genetycznie, co daje możliwość naprawy defektu genetycznego komórek, dalszego ich różnicowania, a w końcu przeszczepienia dawcy.…”