Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins-Reference-Cited by-同舟云学术

Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins

Published:2023-07-07 Issue: Volume: Page:
ISSN:
Container-title:
language:
Short-container-title:

Author:

Blümke Alexander¹,Ijeoma Erica¹,Simon Jessica¹,Wellington Rachel¹,Purwaningrum Medania¹,Doulatov Sergei²,Leber Elizabeth¹,Scatena Marta¹,Giachelli Cecilia M¹^ORCID

Affiliation:

1. University of Washington Department of Bioengineering

2. University of Washington

Abstract

Abstract Background: Ever 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. Methods: In 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. Results: Embryoid-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. Conclusions: The 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.

Publisher

Research Square Platform LLC

Reference113 articles.

1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell [Internet]. 2006 Aug 25 [cited 2023 Mar 24];126(4):663–76. Available from: https://pubmed.ncbi.nlm.nih.gov/16904174/.

2. Singh VK, Kalsan M, Kumar N, Saini A, Chandra R. Induced pluripotent stem cells: Applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev Biol. 2015 Feb 2;3(FEB):2.

3. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007 448:7151 [Internet]. 2007 Jun 6 [cited 2023 Mar 24];448(7151):318–24. Available from: https://www.nature.com/articles/nature05944.

4. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell [Internet]. 2007 Nov 30 [cited 2023 Mar 24];131(5):861–72. Available from: http://www.cell.com/article/S0092867407014717/fulltext.

5. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S et al. Induced pluripotent stem cell lines derived from human somatic cells. Science (1979) [Internet]. 2007 Dec 21 [cited 2023 Mar 24];318(5858):1917–20. Available from: https://www.science.org/doi/10.1126/science.1151526.