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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

Global main effects principal component analysis of GF and SM ACME screen demonstrates that WNT and FGF agonists exert positive effects on T-GFP expression.DOI:http://dx.doi.org/10.7554/eLife.08413.004
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fig1s1: Global main effects principal component analysis of GF and SM ACME screen demonstrates that WNT and FGF agonists exert positive effects on T-GFP expression.DOI:http://dx.doi.org/10.7554/eLife.08413.004

Mentions: (A) Schematic of the ACME experimental design. Human ES cells carrying a green fluorescent protein (GFP) reporter under control of the BRY/T promoter were treated with CHIR98014 (CHR). GFP positive (T-GFP) cells were seeded onto ACME slides printed with combinations of extracellular matrix proteins (ECMPs), growth factors (GF) and small molecules (SMs). A primary screen contained all possible combinations of ECMP Collagen I (C1), Collagen III (C3), Collagen IV (C4), Collagen V (C5), Fibronectin (FN), Laminin (LN), and Vitronectin (VN). A second GF and SM screen contained all possible single, pairwise, and three-way combinations of Wnt3a (WNT), CHIR98014 (CHR), Rspondin (RSP), Dkk-1 (DKK), IWP-2 (IWP), FGF-2 (FGF), FGF-7 (KGF), VEGF (VGF), EGF (EGF), SHH (SHH), Activin (ACT), Cyclopamine (CYC), Dorsomorphin (DSM), BMP4 (BMP), SB4-31542 (SB4), and Noggin (NOG). The second screen was performed on the optimal ECMP combination identified in the primary screen. 72 hr after seeding, GFP expression and DAPI staining were captured and analyzed using a high content imaging microscope. (B) Results of the primary ECMP screen. A heat map of average T-GFP intensity was generated showing the distribution across the data set. Representative clusters are magnified. The position of the Matrigel condition in the cluster is also indicated for reference. Rows represent different ECMP combinations. Columns 1–3 represent biological replicates for cell number (Cell #) or T-GFP (GFP). Columns marked represent the average of the three biological replicates. (C) Representative images of ECMP conditions in the array format. Matrigel is shown in comparison to the hit condition C1 C3 C4 FN VN. Scalebar = 50 µm. (D) Results of the second GF and SM screen. A heat map of average T-GFP intensity was generated showing the distribution across the data set. Representative clusters are magnified. The position of the condition lacking GFs and SMs (No Factor) is also indicated for reference. Rows represent different GF and SM combinations. Columns 1–3 represent biological replicates for cell number (Cell #) or T-GFP (GFP). Columns marked represent the average of the three biological replicates. (E) Representative images of GF and SM conditions in the array format. No GF or SM is shown in comparison to the hit condition CHR + FGF. Scalebar = 50 µm. Figure 1—figure supplement 1 provides a global main effects principal component analysis for all GF and SM used in this second screen.


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)

Global main effects principal component analysis of GF and SM ACME screen demonstrates that WNT and FGF agonists exert positive effects on T-GFP expression.DOI:http://dx.doi.org/10.7554/eLife.08413.004
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631902&req=5

fig1s1: Global main effects principal component analysis of GF and SM ACME screen demonstrates that WNT and FGF agonists exert positive effects on T-GFP expression.DOI:http://dx.doi.org/10.7554/eLife.08413.004
Mentions: (A) Schematic of the ACME experimental design. Human ES cells carrying a green fluorescent protein (GFP) reporter under control of the BRY/T promoter were treated with CHIR98014 (CHR). GFP positive (T-GFP) cells were seeded onto ACME slides printed with combinations of extracellular matrix proteins (ECMPs), growth factors (GF) and small molecules (SMs). A primary screen contained all possible combinations of ECMP Collagen I (C1), Collagen III (C3), Collagen IV (C4), Collagen V (C5), Fibronectin (FN), Laminin (LN), and Vitronectin (VN). A second GF and SM screen contained all possible single, pairwise, and three-way combinations of Wnt3a (WNT), CHIR98014 (CHR), Rspondin (RSP), Dkk-1 (DKK), IWP-2 (IWP), FGF-2 (FGF), FGF-7 (KGF), VEGF (VGF), EGF (EGF), SHH (SHH), Activin (ACT), Cyclopamine (CYC), Dorsomorphin (DSM), BMP4 (BMP), SB4-31542 (SB4), and Noggin (NOG). The second screen was performed on the optimal ECMP combination identified in the primary screen. 72 hr after seeding, GFP expression and DAPI staining were captured and analyzed using a high content imaging microscope. (B) Results of the primary ECMP screen. A heat map of average T-GFP intensity was generated showing the distribution across the data set. Representative clusters are magnified. The position of the Matrigel condition in the cluster is also indicated for reference. Rows represent different ECMP combinations. Columns 1–3 represent biological replicates for cell number (Cell #) or T-GFP (GFP). Columns marked represent the average of the three biological replicates. (C) Representative images of ECMP conditions in the array format. Matrigel is shown in comparison to the hit condition C1 C3 C4 FN VN. Scalebar = 50 µm. (D) Results of the second GF and SM screen. A heat map of average T-GFP intensity was generated showing the distribution across the data set. Representative clusters are magnified. The position of the condition lacking GFs and SMs (No Factor) is also indicated for reference. Rows represent different GF and SM combinations. Columns 1–3 represent biological replicates for cell number (Cell #) or T-GFP (GFP). Columns marked represent the average of the three biological replicates. (E) Representative images of GF and SM conditions in the array format. No GF or SM is shown in comparison to the hit condition CHR + FGF. Scalebar = 50 µm. Figure 1—figure supplement 1 provides a global main effects principal component analysis for all GF and SM used in this second screen.

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