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Development of a simple, repeatable, and cost-effective extracellular matrix for long-term xeno-free and feeder-free self-renewal of human pluripotent stem cells

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Abstract

Given the potential importance of human pluripotent stem cells (hPSCs) in translational research and regenerative medicine, the aim of the present study was to develop a simple, safe, and cost-effective substrate to expand hPSCs. We report the development of an extracellular matrix (ECM), designated “RoGel,” based on conditioned medium (CM) of human fibroblasts under serum- and xeno-free culture conditions. The long-term self-renewal of hPSCs on RoGel was also assessed. The results showed that self-renewal, pluripotency, plating efficiency, and cloning efficiency of hPSCs on this newly developed ECM were similar to those of Matrigel, the conventional mouse-cell line-derived ECM. The cells had the capability to passage mechanically on a cold surface, which resulted in their long-term maintenance with normal karyotype. We have demonstrated that CM-coated plates preserved for 1 year at room temperature maintained the capability of hPSC expansion. This ECM provides an attractive hPSC culture platform for both research and future therapeutic applications.

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Acknowledgments

This study was funded by grants provided by Royan Institute and the Iranian Council of Stem Cell Research and Technology. We appreciate the technical assistance of Ali Akhlaghi, Azam Samadian, Ehsan Janzamin, Fazel Samani, Najmeh Sadat Masoudi, Behrooz Asgari, Ebrahim Shahbazi, and Mostafa Najar.

Conflict of interest

The authors have no conflict of interest to declare.

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Authors and Affiliations

  1. Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Mohammad Pakzad, Mohammad Kazemi Ashtiani & Hossein Baharvand

  2. Department of Biology, Shahed University, Tehran, Iran

    Mohammad Pakzad & Seyed Latif Mousavi-Gargari

  3. Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran

    Hossein Baharvand

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  1. Mohammad Pakzad
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  2. Mohammad Kazemi Ashtiani
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  3. Seyed Latif Mousavi-Gargari
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  4. Hossein Baharvand
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Correspondence to Hossein Baharvand.

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Supplementary material 1 (DOC 33 kb)

418_2013_1144_MOESM2_ESM.tif

Supplementary Figure 1. Feeder cells derived from human dermal fibroblasts (HDFs) and foreskin fibroblasts (HFF) at low and high densities. (TIFF 7,154 kb)

418_2013_1144_MOESM3_ESM.tif

Supplementary Figure 2. Characterization of pluripotency markers and karyotype of hPSCs grown on RoGel. The lines are characterized after five to twenty passages. (A) hPSCs on RoGel retained key properties of pluripotent markers. Morphology of Royan H6 after ten passage and expressions of ALP, OCT4, SSEA3, and TRA-1-81. hiPSC4 and Royan H5 expressed the markers (not shown). Nuclei stained with DAPI (blue). (B) The karyotype of hESCs and hiPSCs after several passages on RoGel was normal. (TIFF 8,331 kb)

418_2013_1144_MOESM4_ESM.tif

Supplementary Figure 3. In vitro and in vivo differentiation of hPSCs grown on RoGel. (A) RT–PCR analysis of in vitro differentiation of hESCs (Royan H6) by EB formation showed the expression of markers of the three embryonic germ layers. (B) Directed differentiation of Royan H6 after 16 passages on RoGel into neural cells in the presence of Noggin, RA, and bFGF. Immunocytofluorescence of the cells showed mature neural markers, TUJ1 in green. Nuclei were stained with propidium iodide (PI) in red. (C) Teratoma formation, macroscopic view of teratoma with distinct retinal pigment epithelium (RPE) on teratoma. Representative photomicrographs showing derivatives from all three germ layers in the teratomas including RPE (ectodermal marker); cartilage (mesodermal marker); and intestinal epithelium (endodermal marker). (TIFF 8,206 kb)

418_2013_1144_MOESM5_ESM.tif

Supplementary Figure 4. Fractionation of RoGel to support self-renewal of hPSCs. (A) The CM fractionated to >10 kDa, <10 kDa, >30 kDa, and <30 kDa by Millipore’s Amicon Ultra-15 centrifugal filter devices based on the molecular weight of CM’s proteins (MWCO). (B) Royan H6 hESCs were cultured on the fractions of CM. Cells grew with hESC morphology when the plates were coated with >10 kDa or >30 kDa fractionated CM. (TIFF 7,062 kb)

418_2013_1144_MOESM6_ESM.tif

Supplementary Figure 5. Phase contrast microscopy images of plates coated with CM or MG. Phase contrast microscopy of air dried- and fresh-coated surfaces of MG and CM in different conditions. The air dried-coated surfaces of CM with >10 kDa and >30 kDa fractions are similar to both air dried MG and CM. (TIFF 12,257 kb)

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Pakzad, M., Ashtiani, M.K., Mousavi-Gargari, S.L. et al. Development of a simple, repeatable, and cost-effective extracellular matrix for long-term xeno-free and feeder-free self-renewal of human pluripotent stem cells. Histochem Cell Biol 140, 635–648 (2013). https://doi.org/10.1007/s00418-013-1144-3

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