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Induction and expansion of human PRRX1+ limb-bud-like mesenchymal cells from pluripotent stem cells

Nature Biomedical Engineering volume 5, pages 926–940 (2021)Cite this article

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Abstract

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1–tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells’ chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.

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Fig. 1: Development of a method to induce PRRX1+ cells using the human PRRX13′tdTomato-reporter iPSC line.
Fig. 2: Development of an expandable culture protocol for LBM-like cells that maintains PRRX1 expression.
Fig. 3: Chondrogenic induction of ExpLBM cells using two- and three-dimensional culture conditions.
Fig. 4: Identification of CD antigens for the prospective assessment of the chondrogenic capacity of ExpLBM cells.
Fig. 5: Recapitulation of a disease phenotype using ExpLBM cells derived from patients with COL2pathy skeletal dysplasia.
Fig. 6: Development of a simple protocol for mass-producing uniform-sized hyaline cartilaginous-like tissues using ExpLBM cells.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and processed RNA-seq data were deposited in the NCBI GEO database under the accession number GSE165620. All data generated in this study, including source data and the data used to make the figures, are available from figshare with the identifier https://doi.org/10.6084/m9.figshare.14888937. Source data are provided with this paper.

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Acknowledgements

We thank N. Tsumaki for providing iPS cell lines derived from patients with COL2pathy (ACGII-1 and HCG-1); M. Hada and M. Nishio for technical assistance with feeder-free cultures; A. Hirao for providing chemical libraries; and K. Sekiguchi, S. Nagata, S. Tamaki, Y. Jin and C. Alev for their invaluable comments and advice. Preparation of slides and staining was supported by the Central Research Laboratory, Okayama University Medical School. We thank the members of the Department of Animal Resources, Advanced Science Research Center and Okayama University for maintaining the mice. This research was supported by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (grant no. 17H04399 to T. Takarada), AMED (grant nos 18bm0704024h0001 and 20bm0404064h0001 to T. Takarada), the Acceleration Program for Intractable Disease Research Utilizing Disease Specific iPS Cells (AMED) to J.T. and the Cooperative Research Program (Joint Usage/Research Center program) of the Institute for Frontier Life and Medical Sciences, Kyoto University to T. Takarada and J.T. These funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Daisuke Yamada, Tomoka Takao, Shota Takihira, Akihiro Miura, Lu Ming & Takeshi Takarada

  2. Precision Health, Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan

    Masahiro Nakamura

  3. Department Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Shota Takihira, Aki Yoshida, Eiji Nakata & Toshifumi Ozaki

  4. Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    Shunsuke Kawai, Hiroyuki Yoshitomi & Junya Toguchida

  5. Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

    Hiroyuki Yoshitomi, Mai Gozu, Kumi Okamoto & Junya Toguchida

  6. Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Hironori Hojo

  7. Institute of Frontier Science and Technology, Okayama University of Science, Okayama, Japan

    Naoyuki Kusaka & Ryosuke Iwai

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Contributions

D.Y. performed the experiments, analysed the data and wrote the manuscript. M.N., T. Takao, S.T., A.Y., S.K., A.M. and L.M. performed the experiments. H.Y., M.G., K.O., N.K. and T. Takarada provided critical materials. H.H., R.I., E.N., T.O. and J.T. discussed the data and provided critical advice. T. Takarada supervised the project and wrote the manuscript.

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Correspondence to Takeshi Takarada.

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The authors declare no competing interests.

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Peer review information Nature Biomedical Engineering thanks Shuibing Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Immunostaining of PRRX1 in human pluripotent stem cells on pluripotency (day 0) or in the PRRX1+ state.

Immunostaining of PRRX1 in hiPSC lines (414C2 and 1383D2) or hESC lines SEES4 and SEES5 at days 0 and 4.

Extended Data Fig. 2 Engraftment of cartilaginous particles in damaged articular cartilage of SCID rats.

A 1-mm drill hole defect was formed in the articular cartilage of SCID rats and then PRRX13´tdTomato-reporter-derived cartilaginous particles were embedded. The knee joints were fixed 4 weeks after transplantation and tissue sections were stained with HE, Toluidine blue or human VIMENTIN. Representative images were shown.

Extended Data Fig. 3 Bone-like-tissue forming capacity of ExpLBM cells.

(a-c) Human bone marrow-derived mesenchymal stem cell (BM-hMSC) or ExpLBM cells were suspended with βTCP and subcutaneously transplanted into NOD-SCID mice. After 8 weeks, mice were sacrificed and tissues were isolated to identify histological differences using HE staining (a), trichrome staining (b) and immunostaining with human VIMENTIN/COLX (c).

Extended Data Fig. 4 Abnormal chondrogenic capacity of ACGII-1- and HCG-1-derived ExpLBM cells.

(a) Alcian blue staining of other ACGII-1 or HCG-1 clone-derived ExpLBM cells after 2DCI. Other clones established from the same patient were induced to ExpLBM cells and their chondrogenic potentials were assessed by Alcian blue staining after 2DCI (n = 3, three biologically independent experiments). (b) Histological analysis of the cartilaginous particles generated using 3DCI. The cartilaginous particles were generated from each ExpLBM cell. Particle sections were stained with HE, Alcian blue or Safranin O or with antibodies against SOX9, COL2, COL1 or COLX. (c) Measurements and comparison of Safranin O-, SOX9-, or COL2-stained areas in Extended Data Fig. 5b (n = 3, three biologically independent experiments). (d) RT–qPCR analysis of the expression of chondrocyte marker genes after 3DCI using cartilaginous particles derived from each ExpLBM cell. All the values were normalized to the ACTB mRNA levels (n = 3, three biologically independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was determined by one-way ANOVA with correction for multiple testing using the Bonferroni method (a, c, d).

Extended Data Fig. 5 Transmission electron microscopy of cartilaginous particles of each ExpLBM cell on day 42 of step 3.

Cartilaginous particles of COL2pathy-ExpLBM cells comprise chondrocytes with a distended endoplasmic reticulum and low abundance of glycogen. Arrows and arrowheads indicate the endoplasmic reticulum (ER) and glycogen, respectively.

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Yamada, D., Nakamura, M., Takao, T. et al. Induction and expansion of human PRRX1+ limb-bud-like mesenchymal cells from pluripotent stem cells. Nat Biomed Eng 5, 926–940 (2021). https://doi.org/10.1038/s41551-021-00778-x

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