Int J Stem Cells.  2019 Nov;12(3):484-496. 10.15283/ijsc19090.

Maintenance of hPSCs under Xeno-Free and Chemically Defined Culture Conditions

• Lim JJ
• Kim HJ
• Rhie BH
• Lee MR
• Choi MJ
• Hong SH
• Kim KS

• KMID: 2465889
• DOI: http://doi.org/10.15283/ijsc19090

Abstract

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.

Keyword

Embryonic stem cells; Induced pluripotent stem cells; Feeder-free; Chemically defined conditions; Extracellular matrices

MeSH Terms

Animals
Cell Culture Techniques
Embryonic Stem Cells
Extracellular Matrix
Feeder Cells
Human Embryonic Stem Cells
Humans
Induced Pluripotent Stem Cells
Pluripotent Stem Cells
Stem Cells

Figure

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  Fig. 1 Application of feeder-free system for hPSCs culture. (A) Phase contrast image of cultured hPSCs maintained in undefined medium on MEF feeder layer with 20% FBS (a) or 20% KSR (b). (B, C) Phase contrast image of cultured hPSCs maintained in chemical defined medium on different kinds of coating matrix (a: Matrigel, b: Vitronectin XF, c: rhLaminin-521 and d: iMatrix511) at passage 1 (B) and 5 (C). All matrix materials were used according to the manufacturer’s instructions. Scale bar, A: 100 μm; B: 200 μm.

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  Fig. 2 hPSCs culture on vitronectin in chemically defined media. Phase contrast image of cultured hPSC maintained on Vitronectin XF in different kinds of chemically defined medium (a: mTeSR1, b: TeSR-E8, c: iPS-Brew XF and d: StemFit) at subculture passages 2 (upper) and 4 (lower). All culture media were used according to the manufacturer’s instructions. All scale bar, 200 μm.

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  Fig. 3 Supplementation of cP1P for improving proliferation of hPSCs. cP1P treatment appears to regulate proliferation of hPSCs by the expansion of total cell populations associated with cell cycle progression. (A) Bright-field (top) and AP staining images (bottom) of hPSC plated on vitronectin-coated plate in the absence (Con) and presence of cP1P (100 mM). All scale bar, 200 μm. On day 7, the hPSC cultures were analyzed for total cell count (B), cell viability test (C) and cell cycle (D). (E) Protein expression of pluripotent markers (REX-1, OCT4, and KLF4) between Con and cP1P-treated group were analyzed by western blot.

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  Fig. 4 hPSC passaging in cell aggregates and single cell suspensions. (A) Before starting to subculture, highly differentiated colonies or the differentiated section of colonies can be removed by swirling or scratching off with a pipette tip under a dissecting microscope. Black arrows indicate the differentiated section of hPSCs. (B, C) Representative bright field (BF) images of hPSCs passaged in cell aggregates (B) and single cell suspensions (C) during cultures. All scale bar, 200 μm.

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  Fig. 5 Characterization of hPSCs cultured in chemically defined medium. (A) Representative BF images of hPSCs grown on vitronectin-coated plate in TeSRTM-E8TM medium for 5 days and typical colony morphology of hPSCs with positive AP staining. (B) Immunofluorescence staining of hPSCs for pluropotency markers (OCT3/4, red; SOX2, green). Cell nuclei were counterstained using DAPI (blue). All scale bars, 100 μm. (C) Flow cytometry analysis of cultured hPSC for REX-1, E-Cadherin, and OCT3/4. (D) hPSCs were differentiated into three germ-layers using the STEMdiffTM Trilineage Differentiation Kit. During differentiation, the shape of each differentiated cell types was clearly different (a: Endoderm, b: Mesoderm and c: Ectoderm lineage). On day 7, the cultured and differentiated-hPSCs were analyzed by immunofluorescence staining (E) and flow cytometry (F). All scale bar, 100 μm. Flow cytometry analysis of differentiated-hPSCs for AFP (endodermal lineage), Brachyury (mesodermal lineage) and MAP2 (ectoderm lineage).

Reference

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