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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

Quality of iPSC cultures after laser-mediated passage. (a) Immunocytochemical analysis of Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 expression immediately following laser-mediated sectioning of iPSC cultures (BIMR L) into 200 μm sections. Hoechst was used as a nuclear counterstain. Note that all markers are expressed homogeneously across iPSC clumps. Scale bar, 1 mm. (b) Immunocytochemical analysis of apoptosis markers, caspase-3, and cleaved PARP, following laser-mediated sectioning of iPSC cultures. Hoechst was used as a nuclear counterstain. Scale bar, 1 mm. (c) Analysis of iPSC (BIMR 6, left) and ESC (H9, right) growth following propagation using laser-mediated passage or collagenase passage. P indicates passage number. Data are shown as mean + s.d. (n = 3).
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fig3: Quality of iPSC cultures after laser-mediated passage. (a) Immunocytochemical analysis of Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 expression immediately following laser-mediated sectioning of iPSC cultures (BIMR L) into 200 μm sections. Hoechst was used as a nuclear counterstain. Note that all markers are expressed homogeneously across iPSC clumps. Scale bar, 1 mm. (b) Immunocytochemical analysis of apoptosis markers, caspase-3, and cleaved PARP, following laser-mediated sectioning of iPSC cultures. Hoechst was used as a nuclear counterstain. Scale bar, 1 mm. (c) Analysis of iPSC (BIMR 6, left) and ESC (H9, right) growth following propagation using laser-mediated passage or collagenase passage. P indicates passage number. Data are shown as mean + s.d. (n = 3).

Mentions: The effect of the laser on human iPSC and ESC quality and pluripotency was examined immediately following laser-mediated sectioning of stem cell cultures into 200 μm sections. As shown in Figure 3(a), pluripotency markers such as Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 were highly expressed in sectioned iPSC cultures. Image analysis demonstrated that all markers were expressed homogeneously across sections, even in cells right next to the laser sectioning lines. In addition, cells right next to the laser cutting lines did not show any significant increase in apoptosis, as measured by immunocytochemical analysis of activated caspase-3 and cleaved PARP four hours after laser-mediated sectioning (Figure 3(b)). Incubation of replicate cultures overnight (i.e., cultures were sectioned into 200 μm sizes and then given fresh medium) resulted in significant growth of cells into the areas previously sectioned using the laser. Morphological and immunocytochemical analysis of these cultures (using the same pluripotency and apoptosis markers above) indicated that cells regrown into the laser sectioning area were indeed undifferentiated human iPSCs. The laser was then used to section a wider area (~1000 μm) into cultures for analysis of growth over several days. Again, these cultures showed no change in morphology, apoptosis, and pluripotency marker expression, indicating that laser processing did not affect stem cell self-renewal or pluripotency (Supplemental Figure 1 of the supplementary material available online at doi:10.1155/2012/926463).


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)

Quality of iPSC cultures after laser-mediated passage. (a) Immunocytochemical analysis of Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 expression immediately following laser-mediated sectioning of iPSC cultures (BIMR L) into 200 μm sections. Hoechst was used as a nuclear counterstain. Note that all markers are expressed homogeneously across iPSC clumps. Scale bar, 1 mm. (b) Immunocytochemical analysis of apoptosis markers, caspase-3, and cleaved PARP, following laser-mediated sectioning of iPSC cultures. Hoechst was used as a nuclear counterstain. Scale bar, 1 mm. (c) Analysis of iPSC (BIMR 6, left) and ESC (H9, right) growth following propagation using laser-mediated passage or collagenase passage. P indicates passage number. Data are shown as mean + s.d. (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Quality of iPSC cultures after laser-mediated passage. (a) Immunocytochemical analysis of Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 expression immediately following laser-mediated sectioning of iPSC cultures (BIMR L) into 200 μm sections. Hoechst was used as a nuclear counterstain. Note that all markers are expressed homogeneously across iPSC clumps. Scale bar, 1 mm. (b) Immunocytochemical analysis of apoptosis markers, caspase-3, and cleaved PARP, following laser-mediated sectioning of iPSC cultures. Hoechst was used as a nuclear counterstain. Scale bar, 1 mm. (c) Analysis of iPSC (BIMR 6, left) and ESC (H9, right) growth following propagation using laser-mediated passage or collagenase passage. P indicates passage number. Data are shown as mean + s.d. (n = 3).
Mentions: The effect of the laser on human iPSC and ESC quality and pluripotency was examined immediately following laser-mediated sectioning of stem cell cultures into 200 μm sections. As shown in Figure 3(a), pluripotency markers such as Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 were highly expressed in sectioned iPSC cultures. Image analysis demonstrated that all markers were expressed homogeneously across sections, even in cells right next to the laser sectioning lines. In addition, cells right next to the laser cutting lines did not show any significant increase in apoptosis, as measured by immunocytochemical analysis of activated caspase-3 and cleaved PARP four hours after laser-mediated sectioning (Figure 3(b)). Incubation of replicate cultures overnight (i.e., cultures were sectioned into 200 μm sizes and then given fresh medium) resulted in significant growth of cells into the areas previously sectioned using the laser. Morphological and immunocytochemical analysis of these cultures (using the same pluripotency and apoptosis markers above) indicated that cells regrown into the laser sectioning area were indeed undifferentiated human iPSCs. The laser was then used to section a wider area (~1000 μm) into cultures for analysis of growth over several days. Again, these cultures showed no change in morphology, apoptosis, and pluripotency marker expression, indicating that laser processing did not affect stem cell self-renewal or pluripotency (Supplemental Figure 1 of the supplementary material available online at doi:10.1155/2012/926463).

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