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Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells.

Du ZW, Chen H, Liu H, Lu J, Qian K, Huang CL, Zhong X, Fan F, Zhang SC - Nat Commun (2015)

Bottom Line: Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics.However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors.More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4).

View Article: PubMed Central - PubMed

Affiliation: Waisman Center, University of Wisconsin, Madison, Wisconsin 53705, USA.

ABSTRACT
Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.

No MeSH data available.


Related in: MedlinePlus

Expansion of OLIG2+ MNPs(A) Representative images of pure OLIG2+ (green)/NKX2.2+ (red) MNPs maintained under different conditions. Scale bars: 50μm. Quantification of OLIG2+/NKX2.2− cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (B) Representative images of Ki67+ (red) proliferating progenitors maintained under different conditions. Scale bars: 50μm. Quantification of Ki67+ cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Schematics showing the expansion of MNPs with the combination of small molecules. (D) Representative images of MNPs expanded for at least 5 passages yet maintained the OLIG2 (green) expression. (E) Cumulative hPSC-derived MNP counts over five passages (passages denoted p1–p5). One 6-well of cells were manually counted in each passage, and total cell numbers were calculated by times passage ratio.
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Figure 2: Expansion of OLIG2+ MNPs(A) Representative images of pure OLIG2+ (green)/NKX2.2+ (red) MNPs maintained under different conditions. Scale bars: 50μm. Quantification of OLIG2+/NKX2.2− cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (B) Representative images of Ki67+ (red) proliferating progenitors maintained under different conditions. Scale bars: 50μm. Quantification of Ki67+ cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Schematics showing the expansion of MNPs with the combination of small molecules. (D) Representative images of MNPs expanded for at least 5 passages yet maintained the OLIG2 (green) expression. (E) Cumulative hPSC-derived MNP counts over five passages (passages denoted p1–p5). One 6-well of cells were manually counted in each passage, and total cell numbers were calculated by times passage ratio.

Mentions: Developmentally, OLIG2+ MNPs are present transiently and they transition to other neuronal (interneuron) and glial (oligodendrocyte) lineages after generation of MNs. For cellular and biochemical analysis, it is crucial to expand the MNPs without losing their ability to produce MNs, which has not been achieved. Since the CHIR+SB+DMH1+RA+Pur condition is highly efficient in specifying and generating MNPs, we asked if the condition could expand the OLIG2+ MNPs in a continuously dividing state. We first examined whether RA and Pur are required for maintaining OLIG2 expression. The MNPs were passaged weekly under the CHIR+SB+DMH1 condition with or without Pur or RA+Pur. After two passages, OLIG2+ MNPs were decreased to 35±5% in the control group (without Pur and RA), to 62±5% in Pur group, and a large population of NKX2.2+ cells appeared in these two groups. In the RA+Pur group, OLIG2+ MNPs were maintained at 91±3% with rare NKX2.2+ cells (Fig. 2A). Therefore, RA and Pur are required for maintaining the identity of MNPs.


Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells.

Du ZW, Chen H, Liu H, Lu J, Qian K, Huang CL, Zhong X, Fan F, Zhang SC - Nat Commun (2015)

Expansion of OLIG2+ MNPs(A) Representative images of pure OLIG2+ (green)/NKX2.2+ (red) MNPs maintained under different conditions. Scale bars: 50μm. Quantification of OLIG2+/NKX2.2− cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (B) Representative images of Ki67+ (red) proliferating progenitors maintained under different conditions. Scale bars: 50μm. Quantification of Ki67+ cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Schematics showing the expansion of MNPs with the combination of small molecules. (D) Representative images of MNPs expanded for at least 5 passages yet maintained the OLIG2 (green) expression. (E) Cumulative hPSC-derived MNP counts over five passages (passages denoted p1–p5). One 6-well of cells were manually counted in each passage, and total cell numbers were calculated by times passage ratio.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4375778&req=5

Figure 2: Expansion of OLIG2+ MNPs(A) Representative images of pure OLIG2+ (green)/NKX2.2+ (red) MNPs maintained under different conditions. Scale bars: 50μm. Quantification of OLIG2+/NKX2.2− cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (B) Representative images of Ki67+ (red) proliferating progenitors maintained under different conditions. Scale bars: 50μm. Quantification of Ki67+ cells is shown on right (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Schematics showing the expansion of MNPs with the combination of small molecules. (D) Representative images of MNPs expanded for at least 5 passages yet maintained the OLIG2 (green) expression. (E) Cumulative hPSC-derived MNP counts over five passages (passages denoted p1–p5). One 6-well of cells were manually counted in each passage, and total cell numbers were calculated by times passage ratio.
Mentions: Developmentally, OLIG2+ MNPs are present transiently and they transition to other neuronal (interneuron) and glial (oligodendrocyte) lineages after generation of MNs. For cellular and biochemical analysis, it is crucial to expand the MNPs without losing their ability to produce MNs, which has not been achieved. Since the CHIR+SB+DMH1+RA+Pur condition is highly efficient in specifying and generating MNPs, we asked if the condition could expand the OLIG2+ MNPs in a continuously dividing state. We first examined whether RA and Pur are required for maintaining OLIG2 expression. The MNPs were passaged weekly under the CHIR+SB+DMH1 condition with or without Pur or RA+Pur. After two passages, OLIG2+ MNPs were decreased to 35±5% in the control group (without Pur and RA), to 62±5% in Pur group, and a large population of NKX2.2+ cells appeared in these two groups. In the RA+Pur group, OLIG2+ MNPs were maintained at 91±3% with rare NKX2.2+ cells (Fig. 2A). Therefore, RA and Pur are required for maintaining the identity of MNPs.

Bottom Line: Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics.However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors.More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4).

View Article: PubMed Central - PubMed

Affiliation: Waisman Center, University of Wisconsin, Madison, Wisconsin 53705, USA.

ABSTRACT
Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.

No MeSH data available.


Related in: MedlinePlus