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Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells.

Kumar N, Richter J, Cutts J, Bush KT, Trujillo C, Nigam SK, Gaasterland T, Brafman D, Willert K - Elife (2015)

Bottom Line: One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential.Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties.Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States.

ABSTRACT
The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.

No MeSH data available.


Related in: MedlinePlus

Validation of high-throughput ACME screens.Scale up analysis of hits from the ACME screens. Human ES cells carrying a GFP reporter under control of the BRY/T promoter were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, GFP positive (T-GFP) cells were cultured in multi-well plates for 72 hr to validate conditions from the ACME screens. (A) GFP+ cells were cultured in multi-well plates coated with 10 hit matrices from the primary ECMP screen as well as Matrigel and a sub-optimal matrix (C1 C4 C5 LN). The optimal matrix (C1 C3 C4 FN VN) was defined as the condition that maintained the highest T-GFP expression and fostered the highest cell number. Statistical comparisons are made to the Matrigel condition. *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (B) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various GF/SM combinations. Statistical comparisons are made to the conditions containing no GF/SM (No Factor). *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (C) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various concentrations of CHR and FGF2 (FGF).DOI:http://dx.doi.org/10.7554/eLife.08413.005
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fig2: Validation of high-throughput ACME screens.Scale up analysis of hits from the ACME screens. Human ES cells carrying a GFP reporter under control of the BRY/T promoter were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, GFP positive (T-GFP) cells were cultured in multi-well plates for 72 hr to validate conditions from the ACME screens. (A) GFP+ cells were cultured in multi-well plates coated with 10 hit matrices from the primary ECMP screen as well as Matrigel and a sub-optimal matrix (C1 C4 C5 LN). The optimal matrix (C1 C3 C4 FN VN) was defined as the condition that maintained the highest T-GFP expression and fostered the highest cell number. Statistical comparisons are made to the Matrigel condition. *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (B) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various GF/SM combinations. Statistical comparisons are made to the conditions containing no GF/SM (No Factor). *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (C) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various concentrations of CHR and FGF2 (FGF).DOI:http://dx.doi.org/10.7554/eLife.08413.005

Mentions: We confirmed the ECMP hit conditions by scaling up the 10 top-performing matrix compositions shown in the heatmap of Figure 1B into traditional cell culture formats. Compared to Matrigel and a sub-optimal matrix (C1 C4 C5 LN), 8 of the 10 ECMP hit conditions significantly increased the percentage of GFP positive cells (Figure 2A). Importantly, in this scaled-up format, the optimal matrix identified in the primary screen (C1 C3 C4 FN VN) consistently led to higher cell numbers and GFP expression compared to the other top ECMP combinations, thus demonstrating the robustness of the ACME screening platform.10.7554/eLife.08413.005Figure 2.Validation of high-throughput ACME screens.


Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells.

Kumar N, Richter J, Cutts J, Bush KT, Trujillo C, Nigam SK, Gaasterland T, Brafman D, Willert K - Elife (2015)

Validation of high-throughput ACME screens.Scale up analysis of hits from the ACME screens. Human ES cells carrying a GFP reporter under control of the BRY/T promoter were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, GFP positive (T-GFP) cells were cultured in multi-well plates for 72 hr to validate conditions from the ACME screens. (A) GFP+ cells were cultured in multi-well plates coated with 10 hit matrices from the primary ECMP screen as well as Matrigel and a sub-optimal matrix (C1 C4 C5 LN). The optimal matrix (C1 C3 C4 FN VN) was defined as the condition that maintained the highest T-GFP expression and fostered the highest cell number. Statistical comparisons are made to the Matrigel condition. *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (B) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various GF/SM combinations. Statistical comparisons are made to the conditions containing no GF/SM (No Factor). *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (C) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various concentrations of CHR and FGF2 (FGF).DOI:http://dx.doi.org/10.7554/eLife.08413.005
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fig2: Validation of high-throughput ACME screens.Scale up analysis of hits from the ACME screens. Human ES cells carrying a GFP reporter under control of the BRY/T promoter were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, GFP positive (T-GFP) cells were cultured in multi-well plates for 72 hr to validate conditions from the ACME screens. (A) GFP+ cells were cultured in multi-well plates coated with 10 hit matrices from the primary ECMP screen as well as Matrigel and a sub-optimal matrix (C1 C4 C5 LN). The optimal matrix (C1 C3 C4 FN VN) was defined as the condition that maintained the highest T-GFP expression and fostered the highest cell number. Statistical comparisons are made to the Matrigel condition. *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (B) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various GF/SM combinations. Statistical comparisons are made to the conditions containing no GF/SM (No Factor). *p < 0.05, **p < 0.005. When p-values are not indicated with * or **, the statistical difference is not significant from the control. (C) GFP+ cells were cultured in multi-well plates coated with the optimal matrix (C1 C3 C4 FN VN) and various concentrations of CHR and FGF2 (FGF).DOI:http://dx.doi.org/10.7554/eLife.08413.005
Mentions: We confirmed the ECMP hit conditions by scaling up the 10 top-performing matrix compositions shown in the heatmap of Figure 1B into traditional cell culture formats. Compared to Matrigel and a sub-optimal matrix (C1 C4 C5 LN), 8 of the 10 ECMP hit conditions significantly increased the percentage of GFP positive cells (Figure 2A). Importantly, in this scaled-up format, the optimal matrix identified in the primary screen (C1 C3 C4 FN VN) consistently led to higher cell numbers and GFP expression compared to the other top ECMP combinations, thus demonstrating the robustness of the ACME screening platform.10.7554/eLife.08413.005Figure 2.Validation of high-throughput ACME screens.

Bottom Line: One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential.Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties.Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States.

ABSTRACT
The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.

No MeSH data available.


Related in: MedlinePlus