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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

Differentiation of IMP cells into metanephric mesenchyme (MM).(A) Schematic of the differentiation protocol. Cells were differentiated in a step-wise manner using the indicated GFs and SMs from undifferentiated ES cells or from IMP cells to IM and subsequently to MM. Stage-specific marker genes expressed during this differentiation process are indicated at the top. ACT = ActivinA, BMP = BMP4, CHR = CHIR98014, d = day, FGF = FGF2, RA = retinoic acid. (B) Upon differentiation towards MM, cells expressed genes associated with kidney lineage. QPCR was performed on ES and IMP cells for the indicated genes at various time points. Fetal kidney RNA (11 gestation weeks) was used as a control. The data is displayed as a heat map with black corresponding to minimal expression and red corresponding to maximal levels. (C–E) IF analysis of MP cell-derived MM. IMP cells were differentiated as depicted in panel A, fixed and stained for the indicated proteins and DNA (DAPI). Numbers refer to percentages of cells expressing the protein of interest. Standard deviation represents the variation between the fields of view used for counting (n = 20). Scale bar = 100 µm.DOI:http://dx.doi.org/10.7554/eLife.08413.020
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fig7: Differentiation of IMP cells into metanephric mesenchyme (MM).(A) Schematic of the differentiation protocol. Cells were differentiated in a step-wise manner using the indicated GFs and SMs from undifferentiated ES cells or from IMP cells to IM and subsequently to MM. Stage-specific marker genes expressed during this differentiation process are indicated at the top. ACT = ActivinA, BMP = BMP4, CHR = CHIR98014, d = day, FGF = FGF2, RA = retinoic acid. (B) Upon differentiation towards MM, cells expressed genes associated with kidney lineage. QPCR was performed on ES and IMP cells for the indicated genes at various time points. Fetal kidney RNA (11 gestation weeks) was used as a control. The data is displayed as a heat map with black corresponding to minimal expression and red corresponding to maximal levels. (C–E) IF analysis of MP cell-derived MM. IMP cells were differentiated as depicted in panel A, fixed and stained for the indicated proteins and DNA (DAPI). Numbers refer to percentages of cells expressing the protein of interest. Standard deviation represents the variation between the fields of view used for counting (n = 20). Scale bar = 100 µm.DOI:http://dx.doi.org/10.7554/eLife.08413.020

Mentions: Since the IMP cells described in this study failed to generate derivatives of LM, we reasoned that these cells may differentiate into cell populations derived from IM, such as kidney and gonads. To test this possibility, we employed a published protocol to differentiate hES cells into renal progenitors (Figure 7A) (Taguchi et al., 2014). This protocol employed several GFs and SMs to promote the differentiation of hES cells to IM and subsequently metanephric mesenchyme (MM). Importantly, IMP cells efficiently acquired gene expression signatures associated with IM and MM as monitored by qPCR (Figure 7B). The gene expression profile of IMP-derived MM exhibited a striking similarity to that of fetal kidney cells. PAX2 and SIX2 were upregulated at day 14 of renal differentiation, indicating commitment to the kidney lineage (Bush et al., 2013). Furthermore, immuno-fluorescence analysis demonstrated that a significant number of cells expressed IM and MM markers PAX2, SALL1, SIX2, WT1 and CDH1 (E-cadherin) (Figure 7C–E). These results suggested that IMP cells, as predicted by the gene expression profile, are restricted to IM and effectively differentiate into cells expressing genes associated with a renal phenotype.10.7554/eLife.08413.020Figure 7.Differentiation of IMP cells into metanephric mesenchyme (MM).


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)

Differentiation of IMP cells into metanephric mesenchyme (MM).(A) Schematic of the differentiation protocol. Cells were differentiated in a step-wise manner using the indicated GFs and SMs from undifferentiated ES cells or from IMP cells to IM and subsequently to MM. Stage-specific marker genes expressed during this differentiation process are indicated at the top. ACT = ActivinA, BMP = BMP4, CHR = CHIR98014, d = day, FGF = FGF2, RA = retinoic acid. (B) Upon differentiation towards MM, cells expressed genes associated with kidney lineage. QPCR was performed on ES and IMP cells for the indicated genes at various time points. Fetal kidney RNA (11 gestation weeks) was used as a control. The data is displayed as a heat map with black corresponding to minimal expression and red corresponding to maximal levels. (C–E) IF analysis of MP cell-derived MM. IMP cells were differentiated as depicted in panel A, fixed and stained for the indicated proteins and DNA (DAPI). Numbers refer to percentages of cells expressing the protein of interest. Standard deviation represents the variation between the fields of view used for counting (n = 20). Scale bar = 100 µm.DOI:http://dx.doi.org/10.7554/eLife.08413.020
© Copyright Policy
Related In: Results  -  Collection

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fig7: Differentiation of IMP cells into metanephric mesenchyme (MM).(A) Schematic of the differentiation protocol. Cells were differentiated in a step-wise manner using the indicated GFs and SMs from undifferentiated ES cells or from IMP cells to IM and subsequently to MM. Stage-specific marker genes expressed during this differentiation process are indicated at the top. ACT = ActivinA, BMP = BMP4, CHR = CHIR98014, d = day, FGF = FGF2, RA = retinoic acid. (B) Upon differentiation towards MM, cells expressed genes associated with kidney lineage. QPCR was performed on ES and IMP cells for the indicated genes at various time points. Fetal kidney RNA (11 gestation weeks) was used as a control. The data is displayed as a heat map with black corresponding to minimal expression and red corresponding to maximal levels. (C–E) IF analysis of MP cell-derived MM. IMP cells were differentiated as depicted in panel A, fixed and stained for the indicated proteins and DNA (DAPI). Numbers refer to percentages of cells expressing the protein of interest. Standard deviation represents the variation between the fields of view used for counting (n = 20). Scale bar = 100 µm.DOI:http://dx.doi.org/10.7554/eLife.08413.020
Mentions: Since the IMP cells described in this study failed to generate derivatives of LM, we reasoned that these cells may differentiate into cell populations derived from IM, such as kidney and gonads. To test this possibility, we employed a published protocol to differentiate hES cells into renal progenitors (Figure 7A) (Taguchi et al., 2014). This protocol employed several GFs and SMs to promote the differentiation of hES cells to IM and subsequently metanephric mesenchyme (MM). Importantly, IMP cells efficiently acquired gene expression signatures associated with IM and MM as monitored by qPCR (Figure 7B). The gene expression profile of IMP-derived MM exhibited a striking similarity to that of fetal kidney cells. PAX2 and SIX2 were upregulated at day 14 of renal differentiation, indicating commitment to the kidney lineage (Bush et al., 2013). Furthermore, immuno-fluorescence analysis demonstrated that a significant number of cells expressed IM and MM markers PAX2, SALL1, SIX2, WT1 and CDH1 (E-cadherin) (Figure 7C–E). These results suggested that IMP cells, as predicted by the gene expression profile, are restricted to IM and effectively differentiate into cells expressing genes associated with a renal phenotype.10.7554/eLife.08413.020Figure 7.Differentiation of IMP cells into metanephric mesenchyme (MM).

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