技术资料

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD,and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs,using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected,cell lines harboring the PSEN2 N141I mutation displayed an increase in the A$\beta$42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit,restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased A$\beta$42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in A$\beta$42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

OBJECTIVE Charcot-Marie-Tooth (CMT) disease is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here,we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. METHODS iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. RESULTS iPSC-derived motor neurons exhibited gene and protein expression,ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient,but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities,including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. INTERPRETATION Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations,including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies. View Publication

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening. View Publication