Subtype-specific neurons obtained from adult humans will be critical to modeling
Subtype-specific neurons obtained from adult humans will be critical to modeling neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). to form NMJs. A chemical screen revealed that the degenerative features of ALS-hiMNs can be remarkably rescued by the small molecule kenpaullone. Taken together our results define a direct and efficient strategy to obtain disease-relevant neuronal subtypes from adult human patients and reveal their promising value in disease modeling and drug identification. motor neuron fate. Complementary DNAs encoding ISL1 and LHX3 were subcloned into a polycistronic lentiviral vector for expression at a 1:1 ratio as this is essential for motor neuron specification (Lee et al. 2012 Primary fibroblasts (Table ASP3026 S1) from three normal (NL) healthy adult humans (AG05811 71 years designated NL1; AG07473 50 years designated NL2; and AG09969 53 years designated NL3) were ASP3026 co-transduced with lentiviruses expressing NGN2-IRES-GFP-T2A-SOX11 and ISL1-T2A-LHX3 (hereafter referred to as NSIL). Two days post viral infection (dpi) these cells were switched to neuron-induction media containing our previously identified extrinsic factors forskolin (FSK) and dorsomorphin (DM) and basic fibroblast growth factor (FGF2) (Liu et al. 2013 Neuronal conversion was monitored daily by live-cell fluorescence microscopy and analyzed by immunocytochemistry at the indicated time points. Remarkably 86 of NSIL virus-transduced adult fibroblasts (indicated by GFP co-expression) were converted to TUBB3+ neuron-like cells by 14 dpi (Figures 1A and 1B). During this conversion process cells rapidly changed their initially flat spread-out morphology to one with bipolar and multipolar processes. They progressively became more elaborate with round or pyramidal somas condensed nuclei long axons and multiple neurites as indicated by specific staining with the pan-neuronal markers MAP2 and NF200 at 21 dpi (Figures 1C and 1D). The converted cells also expressed the presynaptic marker synaptotagmin 1 (SYT1) in a discrete punctate pattern suggesting the establishment of synaptic terminals by 21 dpi in culture (Figure 1E). The inclusion of FSK ASP3026 DM and FGF2 in the culture media is essential for efficient neuronal reprogramming as omission of any small molecule or FGF2 greatly reduced the population of TUBB3+ cells (Figures S1A and S1B). Figure 1 Rapid and efficient conversion of adult human fibroblasts to hiMNs p85 Immunocytochemistry showed that the reprogrammed neurons exclusively expressed markers for spinal motor neurons including HB9 CHAT and VACHT (Figures 1A 1 Over 84% and 95% of TUBB3+ cells co-stained with HB9 and CHAT respectively. In sharp contrast none expressed markers for dopaminergic (TH) or GABAergic (GAD67) neurons. These data indicate that adult human fibroblasts are reprogrammed into spinal motor neurons (hiMNs). qRT-PCR analysis of gene expression showed that hiMNs are a mixture of cervical and/or thoracic spinal motor neurons (Figure S1C). When co-cultured with mouse astrocytes hiMNs survived over 49 dpi outgrew ASP3026 multiple long processes and formed dense neuronal networks throughout the whole culture (Figure 1J). Compared to cells at earlier stages (Figures 1A and 1H) the expression of HB9 is much reduced or diminished by 49 dpi (Figure 1K) resembling its endogenous expression pattern in more mature spinal MNs (Detmer et al. 2008 In contrast hiMNs maintained strong CHAT expression indicative of cholinergic neurotransmitter synthesis (Figure 1K). Direct fate switch without a progenitor stage A time course analysis showed that around 46% and 90% of the virus-transduced cells expressed the mature neuronal marker MAP2 at 7 dpi and 10 dpi respectively (Figure S1D). During this process proliferative neural progenitors were not involved in the NSIL-mediated conversion of adult human fibroblasts. Cell proliferation was examined by 2-hour pulse labeling with 5-bromodeoxyuridine (BrdU) before immunocytochemical ASP3026 analyses at 0 1 3 7 and 10 dpi respectively (Figure S1D and S1E). The non-transduced control cells were efficiently BrdU-labeled under this condition. However none of the converted MAP2+ cells incorporated BrdU when pulsed at 7 or 10 dpi (Figure S1D and S1E). BrdU incorporation appeared to be nontoxic to converted neurons as a majority could be labeled by BrdU if the proliferating fibroblasts were initially treated.