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

Abstract

SUMMARYHuman pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modeling disease processes and developing new therapeutics. However, these applications are hindered by low-efficiency and heterogeneity of target cell types differentiated from hPSCs, such as motor neurons (MNs), as well as our inability to maintain the potency of lineage committed progenitors. Here, by using a combination of small molecules that regulate multiple signaling 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 5 passages so that a single MNP can be amplified to 1×104. This method is reproducible in human induced pluripotent stem cells and is applied to model MNdegenerative diseases and in proof-of-principle drug screening assays.

Canonical and noncanonical Wnt signaling in neural stem/progenitor cells

“…To do so, hES cells, normally cultured as embryoid bodies [115,152], are placed in stringent serum-free culture conditions that selectively facilitate the survival and growth of neural cells [153]. CHIR99021 has been widely used in order to keep cells in an undifferentiated and proliferative state in order to initially increase neural progenitor cell number, and ultimately neuronal yield [16,[154][155][156]. When the cells enter the neural lineage, rosette-like structures appear.…”

“…For example, Wexler et al showed that baseline -catenin signaling represses neuronal differentiation in human NSCs [131], and another study reported that inhibition of Wnt/ -catenin signaling using Dkk-1 or the tankyrase inhibitor XAV939 promotes neural precursor specification [164]. Moreover, GSK-3 inhibition, which induces stem cell renewal and sustains the expression of pluripotency markers [16], is a widely used tool to expand neural progenitors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 [154][155][156], and bFGF treatment of hES and iPS cells, which maintains the undifferentiated state, activates canonical Wnt signaling through inhibition of GSK-3 [165].…”

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

“…Consistently, exogenous activation of Wnt signaling with CHIR99021 increases the expression of posterior markers such as HOXB4, while inhibitor of Wnt-production 2 (IWP2) treatment increases the expression of anterior markers such as FOXG1 [19]. [20] Maury et al, 2015 [22] Therefore, Wnt signaling plays a critical role in the regional patterning and positional identity of hPSC-derived NPCs. Due to the neural patterning effect, Wnt signaling can efficiently promote motor neuron differentiation from hPSCs, in combination with caudalization factor retinoic acid (RA) and ventralization factor SHH.…”

“…Using dual SMAD inhibition in the presence of Wnt activator CHIR99021, high purity of motor neural progenitors (>95% Oligo2 C ) can be generated in 12 days. 20 Consistently, high purity of spinal motor neurons (74% ISL1…”

“…Due to the neural patterning effect, Wnt signaling can efficiently promote motor neuron differentiation from hPSCs, in combination with caudalization factor retinoic acid (RA) and ventralization factor SHH. 20,21 Wnt signaling may elevate the threshold level of SHH signaling to enrich motor neural progenitors. Using dual SMAD inhibition in the presence of Wnt activator CHIR99021, high purity of motor neural progenitors (>95% Oligo2 C ) can be generated in 12 days.…”