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A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells

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ABSTRACT

Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

No MeSH data available.


Comparison of hPSC morphology and growth using the defined, xeno-free hPSC medium.Three hPSC lines were adapted to the defined, xeno-free medium formulation supplemented with either bFGF and heparin or TS bFGF without heparin. Cultures seeded at 2 x 104 cells/cm2, received either daily medium changes with medium containing heparin and bFGF or received medium containing TS bFGF but no heparin every other day (EOD). Scale bar: 200 μm.
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pone.0161229.g003: Comparison of hPSC morphology and growth using the defined, xeno-free hPSC medium.Three hPSC lines were adapted to the defined, xeno-free medium formulation supplemented with either bFGF and heparin or TS bFGF without heparin. Cultures seeded at 2 x 104 cells/cm2, received either daily medium changes with medium containing heparin and bFGF or received medium containing TS bFGF but no heparin every other day (EOD). Scale bar: 200 μm.

Mentions: Finally, to test the ability of TS bFGF to sustain the cultivation of hPSCs in the xeno-free medium formulation, we adapted cells to this new medium and tested whether daily supplementation with fresh hPSC medium containing heparin and bFGF performed better than hPSCs supplemented every other day with hPSC medium containing TS bFGF but no heparin (Fig 3). Using a cell seeding ratio of 2 x 104 cells/cm2, hPSCs cultivated with daily changes of their culture medium reach confluence on day 5 post-seeding. The hPSC morphology and rate of proliferation observed using TS bFGF were consistent with conditions using native bFGF where the culture medium was changed every day. Only minor changes in the compactness of the cells near the periphery of the colony were observed in the hESCs cultivated in the TS bFGF formulation with EOD medium changes. Importantly, no significant changes were observed in the expression of markers of pluripotency, the amount of spontaneous differentiation or in the cells ability to differentiate into cells representing early embryonic ectoderm, mesoderm and endoderm. It should be noted that when bFGF was used in EOD feeding strategy in the presence or absence of heparin, the bFGF level was not sufficient to support PSC growth and induced significant spontaneous differentiation (data not shown).


A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells
Comparison of hPSC morphology and growth using the defined, xeno-free hPSC medium.Three hPSC lines were adapted to the defined, xeno-free medium formulation supplemented with either bFGF and heparin or TS bFGF without heparin. Cultures seeded at 2 x 104 cells/cm2, received either daily medium changes with medium containing heparin and bFGF or received medium containing TS bFGF but no heparin every other day (EOD). Scale bar: 200 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016087&req=5

pone.0161229.g003: Comparison of hPSC morphology and growth using the defined, xeno-free hPSC medium.Three hPSC lines were adapted to the defined, xeno-free medium formulation supplemented with either bFGF and heparin or TS bFGF without heparin. Cultures seeded at 2 x 104 cells/cm2, received either daily medium changes with medium containing heparin and bFGF or received medium containing TS bFGF but no heparin every other day (EOD). Scale bar: 200 μm.
Mentions: Finally, to test the ability of TS bFGF to sustain the cultivation of hPSCs in the xeno-free medium formulation, we adapted cells to this new medium and tested whether daily supplementation with fresh hPSC medium containing heparin and bFGF performed better than hPSCs supplemented every other day with hPSC medium containing TS bFGF but no heparin (Fig 3). Using a cell seeding ratio of 2 x 104 cells/cm2, hPSCs cultivated with daily changes of their culture medium reach confluence on day 5 post-seeding. The hPSC morphology and rate of proliferation observed using TS bFGF were consistent with conditions using native bFGF where the culture medium was changed every day. Only minor changes in the compactness of the cells near the periphery of the colony were observed in the hESCs cultivated in the TS bFGF formulation with EOD medium changes. Importantly, no significant changes were observed in the expression of markers of pluripotency, the amount of spontaneous differentiation or in the cells ability to differentiate into cells representing early embryonic ectoderm, mesoderm and endoderm. It should be noted that when bFGF was used in EOD feeding strategy in the presence or absence of heparin, the bFGF level was not sufficient to support PSC growth and induced significant spontaneous differentiation (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

No MeSH data available.