| Neurodegenerative diseases belong to the type of chronic and progressive neurological disorders and are the major threat to human health. As the population ages, the incidence of neurodegenerative disorders, such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, will further increase. All these neurodegenerative diseases are characterized by gradual deterioration and loss of specific types of neurons in characterized regions within the central nervous system. For example, Parkinson’s disease results from the impairment of dopamine-generating cells in the substantia nigra, a region of the midbrain, and Huntington’s disease is caused by deterioration or death of GABA-ergic projection neurons in the striatum. To date, there are no effective clinical therapies ever developed for these neurodegenerative disorders. Treatments are limited on symptomatic therapies by delivery of neurotransmitters or neurotropic factors. After the death of those damaged neurons, the treatments will no longer be effective. With the development of human pluripotent stem cells, cell transplantation based therapy is now emerging around the corner and remained the only way to combat these devastating diseases.PurposeIn order to fully apply h PSC for studying development and disease, it is crucial to build differentiation protocols for specification of specific types of neuroprogenitors and then neurons. The ideal protocols for lineage specification should be cell type controllable, efficient, chemically-defined and if possible, should faithfully mirror general principles of in vivo development. Embryoid body(EB) formation differentiation paradigm and dual-Smad inhibition-based adherent culture(AD) differentiation paradigm are broadly used for specification of neuroprogenitors and then neurons. h PSC have also been efficiently patterned to medial ganglionic eminence(MGE), lateral ganglionic eminence(LGE) and floor plate(FP). Functional γ-aminobutyric acid(GABA) interneurons, basal forebrain cholinergic neurons, medium spiny GABA neurons as well as midbrain dopamine(DA) neurons are thus efficiently generated from these regional progenitors, which show remarkable therapeutic potentials in correcting behavioral abnormalities resemble of epilepsy, Alzheimer’s disease, Huntington disease and Parkinson’s disease. However, it is largely unknown whether both EB and AD neural differentiation paradigms are suitable for specifying all types of regional neurons or they have any bias.Methods1. The optimized EB and AD differentiation paradigms are used to induce the h PSC into neural progenitors and neurons.2. The h PSC was targeted into different regional neural progenitor with the treatment of SHH and RA under the EB and AD differentiation paradigms.3. The differentiation bias for specifying different types of progenitors and neurons was verified by comprehensive comparation between the EB differentiation paradigm and the AD differentiation paradigm.4. The differences between the EB and AD differentiation paradigms were investigated by m RNA-Seq analyses5. The CRISPR/Cas9 system was used to construct a Foxa2-2A-e GFP reporter line,and small molecule screening experiments were taken by Foxa2 e GFP/W cell line. The effect of selected inhibitors on the cell fate decision was further studied by the differentiation paradigm.Results1. Without exogenous morphogen treatment, neural progenitors specified from both EB and AD paradigms expressed dorsal forebrain genes Foxg1 and Pax6, and the neurons expressed Tuj1, Tbr1 and VGluT1.2. SHH treatment under the EB conditions and the AD conditions induced the differentiation of Pax6+/Nkx2.1- ventral cell fate. Upon RA treatment, Foxg1-/Hoxb4+ hindbrain neural progenitors were induced.3. With the treatment of SHH, EB ventral cells are Nkx2.1+/Sox1+/ Lhx6+/ Lhx8+/Foxa2-/Foxg1+/EN1-, referring the forebrain MGE identity, while AD ventral cells are Nkx2.1+/Sox1-/Lhx6-/Lhx8-/Foxa2+/Foxg1-/EN1+, confirming a FP fate. The neurons were mainly GABA and CHAT positive under EB condition, but TH positive under AD condition.4. The results of m RNA-seq indicated that early developmental, metabolic and adhesion properties predetermine the FP vs MGE specification of AD and EB cells upon SHH patterning.5. The overexpression of Foxg1 or the knockout of Foxa2, Pax6 and Nkx2.1 could not account for the variation of EB and AD differentiation paradigms.6. Small molecule screening experiments by Foxa2 e GFP/W cell line uncover that AG490 and SB202190, the inhibitors of JAK2-STAT3 and p38 MAPK pathways, could switch the FP fate to a MGE fate and then the neuronal fate.ConclusionWe present evidence and show that h PSC mostly take a rostral-dorsal identity during in vitro neural differentiation with both EB and AD paradigms in the absence of patterning morphogens. SHH effectively ventralizes in vitro differentiated human neuroectoderm under both EB and AD conditions to ventral progenitors, as domenstrated by a Pax6 to Nkx2.1 expression shift. However, differential ventral fates are acquired in h ESC under EB vs AD culture conditions in respond to SHH stimulation. A MGE regional fate and then GABA and cholindergic neurons will be generated in SHH treated EB cells, while ventralized AD cells mostly yield FP progenitors and TH+ neurons. Small molecule screening experiments by Foxa2 e GFP/W cell line uncover that JAK2-STAT3 and p38 MAPK pathways may be crucial for proper FP specification and blocking both pathways by AG490 and SB202190 could switch the FP fate to a MGE fate and then the neuronal fate. |
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