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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

MNPs differentiate into enriched functional MNs(A) Schematics showing the time course and small molecule cocktail for MNP differentiation into mature MNs. (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in group). (B) Representative images of MNs showing MNX1+, ISL1+ (green) and CHAT+ (red) on. Scale bars: 50μm. Quantification of MNX1+, ISL1+ and CHAT+ is shown (C) MNs, stained with CHAT antibody (red), formed neuromuscular junctions, labelled with bungarotoxin (BTX, green), when co-cultured with myotubes. Scale bars: 100μm. (D) Representative image of xenotransplantation of GFP labeled human MNs into a developing chicken embryo. Scale bars: 50μm. (D′) magnification of the field showing that human MN axons (GFP+/CHAT+) projected ventrally through the ventral roots.
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Figure 3: MNPs differentiate into enriched functional MNs(A) Schematics showing the time course and small molecule cocktail for MNP differentiation into mature MNs. (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in group). (B) Representative images of MNs showing MNX1+, ISL1+ (green) and CHAT+ (red) on. Scale bars: 50μm. Quantification of MNX1+, ISL1+ and CHAT+ is shown (C) MNs, stained with CHAT antibody (red), formed neuromuscular junctions, labelled with bungarotoxin (BTX, green), when co-cultured with myotubes. Scale bars: 100μm. (D) Representative image of xenotransplantation of GFP labeled human MNs into a developing chicken embryo. Scale bars: 50μm. (D′) magnification of the field showing that human MN axons (GFP+/CHAT+) projected ventrally through the ventral roots.

Mentions: To determine the differentiation of expanded MNPs, we withdrew CHIR+SB+DMH, increased RA concentration (0.5μM), and reduced Pur (0.1μM). After 6 days, nearly all the MNPs differentiated into MNs, as evidenced by expression of MNX1 (90±9%) or ISL1 (95±3%) (Fig. 3A, B). Further culture on Matrigel or astrocyte feeders for two weeks resulted in generation of more mature MNs that expressed CHAT, although the CHAT+ MN population (47±9%) was substantially lower than the MNX1+ MNPs. We reasoned that the lower population of CHAT+ mature MNs may be due to proliferation of the small number of neural precursors and their subsequent differentiation to other neuronal types via lateral inhibition of NOTCH signaling 25. To overcome this inefficiency of MN maturation, we applied Compound E (Cpd E), a NOTCH signaling inhibitor in the MN culture. CpdE treatment for 10 days resulted in a near homogenous MAP2+ mature neuronal cultures without any proliferating cells (Ki67+), and about 91±6% of MAP2+ neurons expressed CHAT (Fig. 3A, B). These CHAT+MNs were electrophysiologically active, as defined by their ability to elicit action potentials in response to depolarizing current injection in current-clamp recordings (Supplementary Fig. 2). Therefore, CpdE not only increases the mature MN population but also substantially shortens the maturation process.


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)

MNPs differentiate into enriched functional MNs(A) Schematics showing the time course and small molecule cocktail for MNP differentiation into mature MNs. (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in group). (B) Representative images of MNs showing MNX1+, ISL1+ (green) and CHAT+ (red) on. Scale bars: 50μm. Quantification of MNX1+, ISL1+ and CHAT+ is shown (C) MNs, stained with CHAT antibody (red), formed neuromuscular junctions, labelled with bungarotoxin (BTX, green), when co-cultured with myotubes. Scale bars: 100μm. (D) Representative image of xenotransplantation of GFP labeled human MNs into a developing chicken embryo. Scale bars: 50μm. (D′) magnification of the field showing that human MN axons (GFP+/CHAT+) projected ventrally through the ventral roots.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: MNPs differentiate into enriched functional MNs(A) Schematics showing the time course and small molecule cocktail for MNP differentiation into mature MNs. (>500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in group). (B) Representative images of MNs showing MNX1+, ISL1+ (green) and CHAT+ (red) on. Scale bars: 50μm. Quantification of MNX1+, ISL1+ and CHAT+ is shown (C) MNs, stained with CHAT antibody (red), formed neuromuscular junctions, labelled with bungarotoxin (BTX, green), when co-cultured with myotubes. Scale bars: 100μm. (D) Representative image of xenotransplantation of GFP labeled human MNs into a developing chicken embryo. Scale bars: 50μm. (D′) magnification of the field showing that human MN axons (GFP+/CHAT+) projected ventrally through the ventral roots.
Mentions: To determine the differentiation of expanded MNPs, we withdrew CHIR+SB+DMH, increased RA concentration (0.5μM), and reduced Pur (0.1μM). After 6 days, nearly all the MNPs differentiated into MNs, as evidenced by expression of MNX1 (90±9%) or ISL1 (95±3%) (Fig. 3A, B). Further culture on Matrigel or astrocyte feeders for two weeks resulted in generation of more mature MNs that expressed CHAT, although the CHAT+ MN population (47±9%) was substantially lower than the MNX1+ MNPs. We reasoned that the lower population of CHAT+ mature MNs may be due to proliferation of the small number of neural precursors and their subsequent differentiation to other neuronal types via lateral inhibition of NOTCH signaling 25. To overcome this inefficiency of MN maturation, we applied Compound E (Cpd E), a NOTCH signaling inhibitor in the MN culture. CpdE treatment for 10 days resulted in a near homogenous MAP2+ mature neuronal cultures without any proliferating cells (Ki67+), and about 91±6% of MAP2+ neurons expressed CHAT (Fig. 3A, B). These CHAT+MNs were electrophysiologically active, as defined by their ability to elicit action potentials in response to depolarizing current injection in current-clamp recordings (Supplementary Fig. 2). Therefore, CpdE not only increases the mature MN population but also substantially shortens the maturation process.

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