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Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

ABSTRACT: BACKGROUND:Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs. METHODS:We selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells). RESULTS:Our results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1?-dependent production and uniform distribution of collagen type I and its interactions with integrins ?2?1 and ?11?1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization. CONCLUSION:The use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

SUBMITTER: Mochizuki M

PROVIDER: S-EPMC7359624 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

Mochizuki Mai M Sagara Hiroshi H Nakahara Taka T

Stem cell research & therapy 20200714 1

<h4>Background</h4>Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on ...[more]

PMID: 32660544

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Project description:BACKGROUND:Currently, ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation. However, because of safety concerns associated with the use of FBS, including potential transmission of zoonotic agents and transplant rejection because of the incorporation of foreign proteins into the stem cells, there is a need for xenogeneic serum-free culture media for clinical handling of stem cells. METHODS:Dental pulp stem cells were derived from wisdom teeth donated by eight healthy volunteers and cultured in xenogeneic serum-free culture medium (XFM) or xenogeneic serum-containing culture medium (SCM). Cells were subjected to morphological, proliferation, karyotype, differentiation, marker expression, cryopreservation, and cytotoxic susceptibility analyses in vitro, as well as transplantation in vivo. RESULTS:In primary culture, XFM cells showed lower adhesion and slightly different morphology, although the single-cell size was similar to that of SCM cells. XFM cells exhibited typical mesenchymal stem cell (MSC) characteristics in vitro and in vivo, including marker gene/protein expression, trilineage differentiation potential, and hard, osteo-dentin tissue formation. Additionally, XFM cells maintained a normal karyotype in vitro and nontumorigenic potential in vivo; however, XFM cells were more susceptible to H2O2 and ultraviolet cytotoxic stimuli. XFM cells formed a multilayered structure showing excessive cell death/division in contrast to the monolayered structure of SCM cells when reaching overconfluence. Proliferation was disrupted in overconfluent XFM cells, and these cells could not be subcultured. Dimethyl sulfoxide-free cryopreserved XFM cells yielded similar results in all of the experiments. CONCLUSIONS:This study is the first reporting successful isolation and expansion of an MSC population from donor-derived tissue (dental pulp) under xenogeneic serum-free culture conditions, as well as the application of cryopreservation, using a research strategy based on clinically oriented in-vitro and in-vivo experiments.

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Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

Publications

Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

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