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Systematic optimization of human pluripotent stem cells media using Design of Experiments.

Marinho PA, Chailangkarn T, Muotri AR - Sci Rep (2015)

Bottom Line: The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming.It also enhances efficient hPSC plating as single cells.Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

View Article: PubMed Central - PubMed

Affiliation: University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, MC 0695, USA.

ABSTRACT
Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

No MeSH data available.


H3K27me3 mark in iPSCs is influenced by culture media. Immunostaining profile of H3K27me3 (puncta) and NANOG and XIST expression on iPSCs derived in different media. Cells derived in iDEAL medium (a, c, d) and in mTeSR1 medium (b, e, f). Immunostaining was performed at the establishment of the line (A, B), passage 4, and after long-term maintenance (c, e), passage 10, with media swap from passages 7 to 10 (d, f). Scale bar, 100 μm. XIST RNA FISH was performed in fibroblast before reprogramming (g), positive control fibroblast (h), iPSC derived in iDEAL (i) and iPSC derived in mTeSR1 (j).
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f5: H3K27me3 mark in iPSCs is influenced by culture media. Immunostaining profile of H3K27me3 (puncta) and NANOG and XIST expression on iPSCs derived in different media. Cells derived in iDEAL medium (a, c, d) and in mTeSR1 medium (b, e, f). Immunostaining was performed at the establishment of the line (A, B), passage 4, and after long-term maintenance (c, e), passage 10, with media swap from passages 7 to 10 (d, f). Scale bar, 100 μm. XIST RNA FISH was performed in fibroblast before reprogramming (g), positive control fibroblast (h), iPSC derived in iDEAL (i) and iPSC derived in mTeSR1 (j).

Mentions: The mechanism of reactivation of X chromosomes during reprogramming is not fully understood23, limiting the potential of iPSCs for X-linked diseases24. It has been shown recently that the addition of recombinant leukemia inhibitory factor (LIF) to the hPSC culture can reactivate inactive X chromosome observed in early passage25, suggesting that media formulations can influence the status of X chromosome. In order to test the hypothesis that iDEAL could influence the X-inactivation status during the derivation of iPSCs, we measured the epigenetic marker trimethylated histone 3 lysine 27 (H3K27me3) in iPSCs derived in mTeSR1 or iDEAL. H3K27me3 marks silenced DNA including the inactive X chromosome in interphase nuclei2627. This mark has been widely used to discriminate the status of X chromosomes in human pluripotent stem cells172829. Half of the iPSC clones derived in iDEAL did not generated H3K27me3 staining foci in early passages (Fig. 5a). However, all iPSC clones derived in mTeSR1 had a very clear positive staining for H3K27me3 (Fig. 5b). Similar results were found for cells after >10 passages thereby ruling out the possibility that iDEAL simply resulted in a delayed transitory state (Fig. 5c). The absence of H3K27me3 marks in late passage of iPSCs could be caused by erosion of XCI24 or by the loss of those marks even though the X chromosome remains inactive30. To test for these possibilities, we performed a media swap experiment and found that the H3K27me3 mark could be detected after changing from iDEAL to mTeSR1 (Fig. 5d). This suggests that the absence of H3K27me3 was not due to either erosion of XCI or the loss of XCI marks in inactive X chromosome. Moreover, these studies allowed us to determine if iDEAL was able to reset this epigenetic marker. Similar to iPSCs derived and maintained in mTeSR1 (Fig. 5e), we found that iPSC clones initially cultured in mTeSR1 retained H3K27me3 foci when transferred to iDEAL (Fig. 5f). This suggests that rather than controlling the presence of H3K27me3, iDEAL maintained the absence status of this marker once it was set upon reprogramming. We validated these observations by performing fluorescent in situ hybridization (FISH) using the XIST RNA expression, another marker for inactive X chromosomes31. Whereas XIST RNA was clearly expressed in fibroblasts before reprogramming (Fig. 5g), positive control (Fig. 5h) and fibroblast-derived iPSCs in mTeSR1 (Fig. 5j), it was not detected in iPSCs derived in iDEAL (Fig. 5i). This result suggests that the medium formulation can affect the X activation status. It is important to note that 3 out of 6 clones from one iPSC line derived in iDEAL showed no H3K27me3 mark for entire population, and one clone showed mixed population for this marker. Nonetheless, our findings, consistent with those of others, demonstrate the importance of culture medium on epigenetic markers involving X inactivation in female iPSCs.


Systematic optimization of human pluripotent stem cells media using Design of Experiments.

Marinho PA, Chailangkarn T, Muotri AR - Sci Rep (2015)

H3K27me3 mark in iPSCs is influenced by culture media. Immunostaining profile of H3K27me3 (puncta) and NANOG and XIST expression on iPSCs derived in different media. Cells derived in iDEAL medium (a, c, d) and in mTeSR1 medium (b, e, f). Immunostaining was performed at the establishment of the line (A, B), passage 4, and after long-term maintenance (c, e), passage 10, with media swap from passages 7 to 10 (d, f). Scale bar, 100 μm. XIST RNA FISH was performed in fibroblast before reprogramming (g), positive control fibroblast (h), iPSC derived in iDEAL (i) and iPSC derived in mTeSR1 (j).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419516&req=5

f5: H3K27me3 mark in iPSCs is influenced by culture media. Immunostaining profile of H3K27me3 (puncta) and NANOG and XIST expression on iPSCs derived in different media. Cells derived in iDEAL medium (a, c, d) and in mTeSR1 medium (b, e, f). Immunostaining was performed at the establishment of the line (A, B), passage 4, and after long-term maintenance (c, e), passage 10, with media swap from passages 7 to 10 (d, f). Scale bar, 100 μm. XIST RNA FISH was performed in fibroblast before reprogramming (g), positive control fibroblast (h), iPSC derived in iDEAL (i) and iPSC derived in mTeSR1 (j).
Mentions: The mechanism of reactivation of X chromosomes during reprogramming is not fully understood23, limiting the potential of iPSCs for X-linked diseases24. It has been shown recently that the addition of recombinant leukemia inhibitory factor (LIF) to the hPSC culture can reactivate inactive X chromosome observed in early passage25, suggesting that media formulations can influence the status of X chromosome. In order to test the hypothesis that iDEAL could influence the X-inactivation status during the derivation of iPSCs, we measured the epigenetic marker trimethylated histone 3 lysine 27 (H3K27me3) in iPSCs derived in mTeSR1 or iDEAL. H3K27me3 marks silenced DNA including the inactive X chromosome in interphase nuclei2627. This mark has been widely used to discriminate the status of X chromosomes in human pluripotent stem cells172829. Half of the iPSC clones derived in iDEAL did not generated H3K27me3 staining foci in early passages (Fig. 5a). However, all iPSC clones derived in mTeSR1 had a very clear positive staining for H3K27me3 (Fig. 5b). Similar results were found for cells after >10 passages thereby ruling out the possibility that iDEAL simply resulted in a delayed transitory state (Fig. 5c). The absence of H3K27me3 marks in late passage of iPSCs could be caused by erosion of XCI24 or by the loss of those marks even though the X chromosome remains inactive30. To test for these possibilities, we performed a media swap experiment and found that the H3K27me3 mark could be detected after changing from iDEAL to mTeSR1 (Fig. 5d). This suggests that the absence of H3K27me3 was not due to either erosion of XCI or the loss of XCI marks in inactive X chromosome. Moreover, these studies allowed us to determine if iDEAL was able to reset this epigenetic marker. Similar to iPSCs derived and maintained in mTeSR1 (Fig. 5e), we found that iPSC clones initially cultured in mTeSR1 retained H3K27me3 foci when transferred to iDEAL (Fig. 5f). This suggests that rather than controlling the presence of H3K27me3, iDEAL maintained the absence status of this marker once it was set upon reprogramming. We validated these observations by performing fluorescent in situ hybridization (FISH) using the XIST RNA expression, another marker for inactive X chromosomes31. Whereas XIST RNA was clearly expressed in fibroblasts before reprogramming (Fig. 5g), positive control (Fig. 5h) and fibroblast-derived iPSCs in mTeSR1 (Fig. 5j), it was not detected in iPSCs derived in iDEAL (Fig. 5i). This result suggests that the medium formulation can affect the X activation status. It is important to note that 3 out of 6 clones from one iPSC line derived in iDEAL showed no H3K27me3 mark for entire population, and one clone showed mixed population for this marker. Nonetheless, our findings, consistent with those of others, demonstrate the importance of culture medium on epigenetic markers involving X inactivation in female iPSCs.

Bottom Line: The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming.It also enhances efficient hPSC plating as single cells.Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

View Article: PubMed Central - PubMed

Affiliation: University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, MC 0695, USA.

ABSTRACT
Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

No MeSH data available.