Dataset Information

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 hu?man 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-?3 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-?3. 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.

SUBMITTER: Suchorska WM

PROVIDER: S-EPMC5380716 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Suchorska Wiktoria Maria WM Augustyniak Ewelina E Richter Magdalena M Trzeciak Tomasz T

Stem cell reviews and reports 20170401 2

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 pr ...[more]

PMID: 27987073

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Project description:BackgroundEver since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferrable regarding the differentiation of osteoclasts.MethodsIn this study we compare the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. Results were validated using qRT-PCR throughout the differentiation stages.ResultsEmbryoid-body based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts.ConclusionsThe outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.

Project description:BackgroundEver since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferable regarding the differentiation of osteoclasts.MethodsIn this study, we compared the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. The results were validated using qRT-PCR throughout the differentiation stages.ResultsEmbryoid body-based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts.ConclusionsThe outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.

Dataset Information

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

Publications

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

Similar Datasets

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