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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.


The optimized iDEAL medium increases pluripotency of hPSC. (a) Representative images of iPSCs passage 20 derived in iDEAL or mTeSR1 when the media were changed daily (top), or skipped on day 5 and 6 (bottom). Scale bar, 200 μm. (b) Representative cell cycle profiles of iPSC assessed by flow cytometry at day 7 after passaging when media were changed daily. (c) Sub-G1 population (%) at day 7 after passaging when media were changed daily. (d) Representative expression of pluripotent markers OCT4 and NANOG assessed by flow cytometry at day 7 after passaging when media were changed daily. (e) iPSC population (%) with OCT4 and NANOG expression at day 7 after passaging when media were changed daily. (f) Representative cell cycle profiles of iPSC at day 7 after passaging when media were skipped on day 5 and 6. (g) Change in sub-G1 population at day 7 after passaging when media were skipped on day 5 and 6. For each media type, sub-G1 iPSC population (%) in skipped condition was normalized to that in daily condition. (h) Representative expression of OCT4 and NANOG at day 7 after passaging when media were skipped on day 5 and 6. (i) Change in OCT4+and NANOG+population at day 7 after passaging when media change was skipped on day 5 and 6. For each media type, iPSC population (%) with OCT4 and NANOG expression in skipped condition was normalized to that in daily condition. Black bars represent iPSC derived in iDEAL, White bars represent iPSC derived in mTeSR1. For (c,e,g,i), data were shown as mean ± s.e.m for each of 3 iPSC lines (one clone each). n (technical replicates)=3. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided unpaired Student’s t test.
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f3: The optimized iDEAL medium increases pluripotency of hPSC. (a) Representative images of iPSCs passage 20 derived in iDEAL or mTeSR1 when the media were changed daily (top), or skipped on day 5 and 6 (bottom). Scale bar, 200 μm. (b) Representative cell cycle profiles of iPSC assessed by flow cytometry at day 7 after passaging when media were changed daily. (c) Sub-G1 population (%) at day 7 after passaging when media were changed daily. (d) Representative expression of pluripotent markers OCT4 and NANOG assessed by flow cytometry at day 7 after passaging when media were changed daily. (e) iPSC population (%) with OCT4 and NANOG expression at day 7 after passaging when media were changed daily. (f) Representative cell cycle profiles of iPSC at day 7 after passaging when media were skipped on day 5 and 6. (g) Change in sub-G1 population at day 7 after passaging when media were skipped on day 5 and 6. For each media type, sub-G1 iPSC population (%) in skipped condition was normalized to that in daily condition. (h) Representative expression of OCT4 and NANOG at day 7 after passaging when media were skipped on day 5 and 6. (i) Change in OCT4+and NANOG+population at day 7 after passaging when media change was skipped on day 5 and 6. For each media type, iPSC population (%) with OCT4 and NANOG expression in skipped condition was normalized to that in daily condition. Black bars represent iPSC derived in iDEAL, White bars represent iPSC derived in mTeSR1. For (c,e,g,i), data were shown as mean ± s.e.m for each of 3 iPSC lines (one clone each). n (technical replicates)=3. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided unpaired Student’s t test.

Mentions: To determine whether iDEAL improves the maintenance of pluripotency in hPSCs, we generated 3 lines of induced pluripotent stem cells (iPSC1, iPSC2 and iPSC3) from isolated female human fibroblasts using the methods described elsewhere17. After manual isolation from mouse embryonic fibroblasts (MEFs), iPSCs were cultivated without feeder cells using iDEAL or mTeSR1 as culture medium and Matrigel as extracellular matrix. Number of iPSC clones and technical replicates for each experiment were summarized in Supplementary Table 3. Similar to mTeSR1, all iPSCs derived in iDEAL expressed pluripotent markers, were able to differentiate into 3 germ layers in vitro and in vivo (teratomas), and maintained a normal karyotype (Supplementary Fig. 1). Late passage karyotyping analyses were also performed in different cell lines, as shown in our published patent16. After 7 days in culture with media changed on a daily basis, iPSCs (passage 20) derived in iDEAL or mTeSR1 are morphologically identical (Fig. 3a, top row) and have a similar percentage of cells undergoing apoptosis (sub-G1 phase population) as assessed by propidium iodide staining followed by flow cytometry1819 (Fig. 3b,c). Regardless of the slightly intrinsic variations of different G1, S and G2/M populations between lines (Supplementary Fig. 2), it is undeniable that iDEAL maintained pluripotency better than mTeSR1, as indicated by a significantly (P = 0.0035 for iPSC1, P = 0.0033 for iPSC2 and P < 0.0001 for iPSC3, two-sided unpaired Student’s t test) higher percentage of double OCT4- and NANOG-positive cells (Fig. 3d,e). It was notable that even when the media was changed daily, approximately 60–70% of the cells remained pluripotent when cultured in mTeSR1, a finding we attribute to the result of unbiased passaging where differentiated cells are not removed prior to passaging (see Methods). In contrast, iDEAL cultures did not accumulate these differentiated cells.


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

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

The optimized iDEAL medium increases pluripotency of hPSC. (a) Representative images of iPSCs passage 20 derived in iDEAL or mTeSR1 when the media were changed daily (top), or skipped on day 5 and 6 (bottom). Scale bar, 200 μm. (b) Representative cell cycle profiles of iPSC assessed by flow cytometry at day 7 after passaging when media were changed daily. (c) Sub-G1 population (%) at day 7 after passaging when media were changed daily. (d) Representative expression of pluripotent markers OCT4 and NANOG assessed by flow cytometry at day 7 after passaging when media were changed daily. (e) iPSC population (%) with OCT4 and NANOG expression at day 7 after passaging when media were changed daily. (f) Representative cell cycle profiles of iPSC at day 7 after passaging when media were skipped on day 5 and 6. (g) Change in sub-G1 population at day 7 after passaging when media were skipped on day 5 and 6. For each media type, sub-G1 iPSC population (%) in skipped condition was normalized to that in daily condition. (h) Representative expression of OCT4 and NANOG at day 7 after passaging when media were skipped on day 5 and 6. (i) Change in OCT4+and NANOG+population at day 7 after passaging when media change was skipped on day 5 and 6. For each media type, iPSC population (%) with OCT4 and NANOG expression in skipped condition was normalized to that in daily condition. Black bars represent iPSC derived in iDEAL, White bars represent iPSC derived in mTeSR1. For (c,e,g,i), data were shown as mean ± s.e.m for each of 3 iPSC lines (one clone each). n (technical replicates)=3. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided unpaired Student’s t test.
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Related In: Results  -  Collection

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f3: The optimized iDEAL medium increases pluripotency of hPSC. (a) Representative images of iPSCs passage 20 derived in iDEAL or mTeSR1 when the media were changed daily (top), or skipped on day 5 and 6 (bottom). Scale bar, 200 μm. (b) Representative cell cycle profiles of iPSC assessed by flow cytometry at day 7 after passaging when media were changed daily. (c) Sub-G1 population (%) at day 7 after passaging when media were changed daily. (d) Representative expression of pluripotent markers OCT4 and NANOG assessed by flow cytometry at day 7 after passaging when media were changed daily. (e) iPSC population (%) with OCT4 and NANOG expression at day 7 after passaging when media were changed daily. (f) Representative cell cycle profiles of iPSC at day 7 after passaging when media were skipped on day 5 and 6. (g) Change in sub-G1 population at day 7 after passaging when media were skipped on day 5 and 6. For each media type, sub-G1 iPSC population (%) in skipped condition was normalized to that in daily condition. (h) Representative expression of OCT4 and NANOG at day 7 after passaging when media were skipped on day 5 and 6. (i) Change in OCT4+and NANOG+population at day 7 after passaging when media change was skipped on day 5 and 6. For each media type, iPSC population (%) with OCT4 and NANOG expression in skipped condition was normalized to that in daily condition. Black bars represent iPSC derived in iDEAL, White bars represent iPSC derived in mTeSR1. For (c,e,g,i), data were shown as mean ± s.e.m for each of 3 iPSC lines (one clone each). n (technical replicates)=3. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided unpaired Student’s t test.
Mentions: To determine whether iDEAL improves the maintenance of pluripotency in hPSCs, we generated 3 lines of induced pluripotent stem cells (iPSC1, iPSC2 and iPSC3) from isolated female human fibroblasts using the methods described elsewhere17. After manual isolation from mouse embryonic fibroblasts (MEFs), iPSCs were cultivated without feeder cells using iDEAL or mTeSR1 as culture medium and Matrigel as extracellular matrix. Number of iPSC clones and technical replicates for each experiment were summarized in Supplementary Table 3. Similar to mTeSR1, all iPSCs derived in iDEAL expressed pluripotent markers, were able to differentiate into 3 germ layers in vitro and in vivo (teratomas), and maintained a normal karyotype (Supplementary Fig. 1). Late passage karyotyping analyses were also performed in different cell lines, as shown in our published patent16. After 7 days in culture with media changed on a daily basis, iPSCs (passage 20) derived in iDEAL or mTeSR1 are morphologically identical (Fig. 3a, top row) and have a similar percentage of cells undergoing apoptosis (sub-G1 phase population) as assessed by propidium iodide staining followed by flow cytometry1819 (Fig. 3b,c). Regardless of the slightly intrinsic variations of different G1, S and G2/M populations between lines (Supplementary Fig. 2), it is undeniable that iDEAL maintained pluripotency better than mTeSR1, as indicated by a significantly (P = 0.0035 for iPSC1, P = 0.0033 for iPSC2 and P < 0.0001 for iPSC3, two-sided unpaired Student’s t test) higher percentage of double OCT4- and NANOG-positive cells (Fig. 3d,e). It was notable that even when the media was changed daily, approximately 60–70% of the cells remained pluripotent when cultured in mTeSR1, a finding we attribute to the result of unbiased passaging where differentiated cells are not removed prior to passaging (see Methods). In contrast, iDEAL cultures did not accumulate these differentiated cells.

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.