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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

Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μm. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test, with a P value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.
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fig4: Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μm. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test, with a P value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.

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)

Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μm. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test, with a P value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μm. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using t-test, with a P value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.
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