Summary

A Novel Culture Modèle de cellules souches pluripotentes humaines Propagation de gélatine dans les médias Placenta conditionné

Published: August 03, 2015
doi:

Summary

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

Abstract

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

Introduction

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.

Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.

The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed.

This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

Protocol

Déclaration éthique: L'étude de placenta humain a été réalisée de manière prospective, avec l'approbation de l'Institutional Review Board pour la recherche humaine de l'Université de Corée (AN09085-001). Toutes les expériences ont été réalisées dans une salle blanche Germ-gratuit au Centre médical de l'Université de Corée. La conception et les procédures utilisant hPSCs expérimentale ont été approuvés par l'Institutional Review Board de l'University Medical Center Coré…

Representative Results

Un avantage de cette méthode de propagation de HPSC est qu'elle utilise des composants sécrétées à partir de cellules humaines. Une étape la plus ciritical dans le protocole est l'isolement et la culture de cellules placentaires humaines dérivées. Cela exige et la dissection précise d'une partie précise de HPC plaque villosités. La figure 1 montre la procédure pour l'isolement cellulaire dérivée du placenta humain. Ce procédé est simple et commode pour l'isolement de …

Discussion

Ce modèle a été mis au point pour se propager avec succès hPSCs, tout en conservant leurs caractéristiques, dans des conditions de culture sans cellules nourricières humanisés sans l'addition de facteurs de croissance recombinants exogènes tels que bFGF ou de l'insuline, ce qui permet la manipulation de hPSCs dans un micro-environnement humanisé. Exogène supplémentation bFGF est commun et l'application de substrats tels que Matrigel ou laminine est essentiel pour la culture libre-chargeur de hPSCs…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Les auteurs tiennent à remercier Soon-Cheol Hong (MD, Ph.D., professeur agrégé, Département d'obstétrique et de gynécologie, Collège médical de l'Université de Corée) pour fournir le tissu placentaire. Ce travail a été financé en partie par des subventions (R1211902) de la National Research Foundation de Corée (NRF), République de Corée.

Materials

Mitomycin-C Sigma-Aldrich Corporation M4287 10ug/ml
Mycoplasma detection kit TaKaRa Bio Inc. #6601
Matrigel BD Biosciences #354277
mTeSR1 STEMCELL Technologies Inc. #05850
Dispase Worthington Biochemical Corporation LS02100 1mg/ml
Gelatin Sigma-Aldrich Corporation G2500 0.10%
BM-Cyclin Roche 799 050 10ug/ml
RNeasy mini kit  Qiagen 74104
Nano Drop Spectrophotometer Thermo Fisher Scientific Inc
iQ SYBR Green qPCR Master Mix  Bio-Rad Laboratories #170-8882AP
ES Cell Characterization kit Chemicon International, Inc., SCR001
Power cDNA Synthesis Kit  iNtRON Biotechnology 25011
QIAamp® DNA Micro kit Qiagen 56304
AmpF/STR® Identifiler® PCR Amplification kit Applied Biosystems Inc. 4322288
Applied Biosystems® 3130xl Genetic Analyzer  Applied Biosystems Inc.

References

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Cite This Article
Jung, J., Kim, B. S. A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media. J. Vis. Exp. (102), e53204, doi:10.3791/53204 (2015).

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