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Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Hohenstein Elliott KA, Peterson C, Soundararajan A, Kan N, Nelson B, Spiering S, Mercola M, Bright GR - Stem Cells Int (2012)

Bottom Line: Standardization is critical for all future applications of stem cells and necessary to fully understand their potential.This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines.Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Intrexon Corporation, Cell Engineering Unit, 6620 Mesa Ridge Road, San Diego, CA 92121, USA.

ABSTRACT
Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

No MeSH data available.


Related in: MedlinePlus

iPSCs propagated by laser-mediated passage differentiated more efficiently into cardiomyocytes. (a) Brightfield image of day 4 EBs generated from iPSC cultures (BIMR A) propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. Scale bar, 250 μm. (b) Size of EBs generated from iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment (n = 35 EBs per data point). Data are shown as scatter plot with red line indicating mean and CV shown in red text above each sample. The asterisks (*) indicate variances that are statistically significant when compared to laser using ANOVA, with P ≤ 0.05 considered significant. (c) Percentage of EBs containing contracting areas. Data are shown as mean + s.d. (n = 2 independent experiments containing 75 EBs/sample in each experiment). (d) QRT-PCR analysis of cardiomyocyte-associated gene expression in EBs generated using iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. The asterisks (*) indicate values that are statistically significant as compared with EBs generated from collagenase passaged iPSC cultures. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test with P ≤ 0.05 considered significant. (e) Expression of cardiomyocyte markers, α-MHC and α-actinin, in EBs generated from iPSC cultures propagated by laser-mediated passage or collagenase treatment on day 22 of cardiac differentiation. Hoechst was used as a nuclear counterstain. Scale bars, 250 μm.
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fig5: iPSCs propagated by laser-mediated passage differentiated more efficiently into cardiomyocytes. (a) Brightfield image of day 4 EBs generated from iPSC cultures (BIMR A) propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. Scale bar, 250 μm. (b) Size of EBs generated from iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment (n = 35 EBs per data point). Data are shown as scatter plot with red line indicating mean and CV shown in red text above each sample. The asterisks (*) indicate variances that are statistically significant when compared to laser using ANOVA, with P ≤ 0.05 considered significant. (c) Percentage of EBs containing contracting areas. Data are shown as mean + s.d. (n = 2 independent experiments containing 75 EBs/sample in each experiment). (d) QRT-PCR analysis of cardiomyocyte-associated gene expression in EBs generated using iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. The asterisks (*) indicate values that are statistically significant as compared with EBs generated from collagenase passaged iPSC cultures. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test with P ≤ 0.05 considered significant. (e) Expression of cardiomyocyte markers, α-MHC and α-actinin, in EBs generated from iPSC cultures propagated by laser-mediated passage or collagenase treatment on day 22 of cardiac differentiation. Hoechst was used as a nuclear counterstain. Scale bars, 250 μm.

Mentions: Statistical analyses were performed using GraphPad Prism with a P value of ≤0.05 considered to be significant. One way analysis of variance (ANOVA) with Bartlett's test for equal variances was performed to evaluate resulting colony sizes generated after passage by five techniques (n = 20 colonies/sample, Figure 2(c)) and resulting EB sizes generated from laser-mediated, collagenase, and trypsin-passaged cells (n = 30 EB/sample, Figure 5(b)). Statistical analysis of QRT-PCR data (n = 3, Figures 4(c) and 5(d)) was performed using a two-tailed t-test.


Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Hohenstein Elliott KA, Peterson C, Soundararajan A, Kan N, Nelson B, Spiering S, Mercola M, Bright GR - Stem Cells Int (2012)

iPSCs propagated by laser-mediated passage differentiated more efficiently into cardiomyocytes. (a) Brightfield image of day 4 EBs generated from iPSC cultures (BIMR A) propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. Scale bar, 250 μm. (b) Size of EBs generated from iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment (n = 35 EBs per data point). Data are shown as scatter plot with red line indicating mean and CV shown in red text above each sample. The asterisks (*) indicate variances that are statistically significant when compared to laser using ANOVA, with P ≤ 0.05 considered significant. (c) Percentage of EBs containing contracting areas. Data are shown as mean + s.d. (n = 2 independent experiments containing 75 EBs/sample in each experiment). (d) QRT-PCR analysis of cardiomyocyte-associated gene expression in EBs generated using iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. The asterisks (*) indicate values that are statistically significant as compared with EBs generated from collagenase passaged iPSC cultures. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test with P ≤ 0.05 considered significant. (e) Expression of cardiomyocyte markers, α-MHC and α-actinin, in EBs generated from iPSC cultures propagated by laser-mediated passage or collagenase treatment on day 22 of cardiac differentiation. Hoechst was used as a nuclear counterstain. Scale bars, 250 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: iPSCs propagated by laser-mediated passage differentiated more efficiently into cardiomyocytes. (a) Brightfield image of day 4 EBs generated from iPSC cultures (BIMR A) propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. Scale bar, 250 μm. (b) Size of EBs generated from iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment (n = 35 EBs per data point). Data are shown as scatter plot with red line indicating mean and CV shown in red text above each sample. The asterisks (*) indicate variances that are statistically significant when compared to laser using ANOVA, with P ≤ 0.05 considered significant. (c) Percentage of EBs containing contracting areas. Data are shown as mean + s.d. (n = 2 independent experiments containing 75 EBs/sample in each experiment). (d) QRT-PCR analysis of cardiomyocyte-associated gene expression in EBs generated using iPSC cultures propagated by laser-mediated passage, trypsin dissociation, or collagenase treatment. The asterisks (*) indicate values that are statistically significant as compared with EBs generated from collagenase passaged iPSC cultures. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test with P ≤ 0.05 considered significant. (e) Expression of cardiomyocyte markers, α-MHC and α-actinin, in EBs generated from iPSC cultures propagated by laser-mediated passage or collagenase treatment on day 22 of cardiac differentiation. Hoechst was used as a nuclear counterstain. Scale bars, 250 μm.
Mentions: Statistical analyses were performed using GraphPad Prism with a P value of ≤0.05 considered to be significant. One way analysis of variance (ANOVA) with Bartlett's test for equal variances was performed to evaluate resulting colony sizes generated after passage by five techniques (n = 20 colonies/sample, Figure 2(c)) and resulting EB sizes generated from laser-mediated, collagenase, and trypsin-passaged cells (n = 30 EB/sample, Figure 5(b)). Statistical analysis of QRT-PCR data (n = 3, Figures 4(c) and 5(d)) was performed using a two-tailed t-test.

Bottom Line: Standardization is critical for all future applications of stem cells and necessary to fully understand their potential.This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines.Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Intrexon Corporation, Cell Engineering Unit, 6620 Mesa Ridge Road, San Diego, CA 92121, USA.

ABSTRACT
Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

No MeSH data available.


Related in: MedlinePlus