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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

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.


hPSCs cultured for over 40 passages using the defined, xeno-free hPSC medium differentiate readily to early ectoderm, mesoderm and endoderm (EB formation).A) Phase contrast images of the hPSC lines just prior to differentiation. B) Embryoid body formation in cultures differentiated for 14 days in culture. C) Antibodies detecting Beta-III-Tubulin (TUJ1) are shown in red; Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown in green. Nuclei were visualized using DAPI (blue). Scale bar: 200 μm.
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pone.0161229.g006: hPSCs cultured for over 40 passages using the defined, xeno-free hPSC medium differentiate readily to early ectoderm, mesoderm and endoderm (EB formation).A) Phase contrast images of the hPSC lines just prior to differentiation. B) Embryoid body formation in cultures differentiated for 14 days in culture. C) Antibodies detecting Beta-III-Tubulin (TUJ1) are shown in red; Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown in green. Nuclei were visualized using DAPI (blue). Scale bar: 200 μm.

Mentions: To further evaluate the pluripotency of the cell lines cultivated in this defined medium, we assessed their ability to differentiate in vitro. After 40 passages, the hPSC lines readily formed embryoid bodies (EBs; Fig 6). Immunocytochemical analysis confirmed the presence of cells expressing class III beta tubulin (Tuj1), smooth muscle actin (SMA), and alpha-feto protein (AFP) representing markers of early embryonic ectoderm, mesoderm and endoderm, respectively. Moreover, teratoma formation analysis demonstrated that both the hESC and hiPSC lines displayed classical histological features of ectoderm, mesoderm and endoderm (S1 Fig).


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
hPSCs cultured for over 40 passages using the defined, xeno-free hPSC medium differentiate readily to early ectoderm, mesoderm and endoderm (EB formation).A) Phase contrast images of the hPSC lines just prior to differentiation. B) Embryoid body formation in cultures differentiated for 14 days in culture. C) Antibodies detecting Beta-III-Tubulin (TUJ1) are shown in red; Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown in green. Nuclei were visualized using DAPI (blue). Scale bar: 200 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0161229.g006: hPSCs cultured for over 40 passages using the defined, xeno-free hPSC medium differentiate readily to early ectoderm, mesoderm and endoderm (EB formation).A) Phase contrast images of the hPSC lines just prior to differentiation. B) Embryoid body formation in cultures differentiated for 14 days in culture. C) Antibodies detecting Beta-III-Tubulin (TUJ1) are shown in red; Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown in green. Nuclei were visualized using DAPI (blue). Scale bar: 200 μm.
Mentions: To further evaluate the pluripotency of the cell lines cultivated in this defined medium, we assessed their ability to differentiate in vitro. After 40 passages, the hPSC lines readily formed embryoid bodies (EBs; Fig 6). Immunocytochemical analysis confirmed the presence of cells expressing class III beta tubulin (Tuj1), smooth muscle actin (SMA), and alpha-feto protein (AFP) representing markers of early embryonic ectoderm, mesoderm and endoderm, respectively. Moreover, teratoma formation analysis demonstrated that both the hESC and hiPSC lines displayed classical histological features of ectoderm, mesoderm and endoderm (S1 Fig).

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.