Human pluripotent stem cells

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below. 1. Analyzed datasets from naive-like growth conditions for pluripotent stem cells: One of the most important properties of human pluripotent stem cells (hPSCs) is related to their ground or naive pluripotent state, which may have major impacts on hPSC growth, genetic engineering, disease modeling, and drug discovery. The Unit has derived and comprehensively characterized naive-like hPSCs (NLPs) using various normoxic and hypoxic conditions. Our comparative meta-analysis indicates the existence heterogeneous pluripotent states in diverse NLPs generated from different naive protocols. Interestingly, some NLPs exhibit much lower single cell plating efficiency, commonly lacking unique mouse and human NLP marker expression. These cells represent an unrecognized minimal naive-like state downstream of formative pluripotency and a trivalent metabolome. Moreover, we revealed a unique metabolome associated with a limited metabolic reprograming capacity in these cells. Our current data provide significant insights into pluripotent state transitions and their associated downstream lineage priming. A manuscript is under revision. 2. Disease model generation: Sjogren's syndrome: Sjogrens syndrome is a disorder of the immune system with symptoms of dry eyes and dry mouth. To construct animal models for Sjogrens syndrome in vitro, embryonic fibroblasts from mouse models for Sjogrens syndrome (Bgn KO and Gol6a2 KO) as well as wildtype control mouse were reprogrammed to induced pluripotent stem cells, in collaboration with Dr. Youngmi Ji, NIDCR. Hypopituitarism: In collaboration with Elyse Moore and Dr. Prashant Chittiboina, NINDS, we study pituitary adenomas including ones that cause Cushing's disease. Ongoing work includes generating and maintaining hiPSCs cultures, creating reporter lines, formulating various differentiation protocols to increase the efficiency of generating hormone-producing cells, and characterizing cellular properties of differentiated cells with immunofluorescence. We successfully differentiated hiPSCs into mature and functional corticotropic cells with the ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Batten disease: In collaboration with Dr. An Dang Do and Dr. Forbes Porter, NICHD, we study Batten disease that has an estimated prevalence of 1:100,000. It is a fatal disease of the nervous system that typically begins in childhood. One subtype of Batten disease, called juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by the mutations in the CLN3 gene which encodes Battenin. We developed human iPSC lines made from patient fibroblasts with various CLN3 mutations. Most of the lines are compound heterozygotes. As controls for assessing the effect of the mutations in each cell line, gene-corrected isogenic lines were generated for each mutation as well as both mutations in each hiPSC line, using CRISPR-cas9-assisted genome modification. So far, 12 different isogenic control lines have been generated. Madras motor neuron disease: MMND is characterized by weakness and atrophy of limbs, multiple lower cranial nerve palsies and sensorineural hearing loss. To study the pathophysiology of the MMND, skin fibroblasts from two patients suffering from MMND were reprogrammed to hiPSC lines using Sendai virus. in collaboration with Dr. Christopher Grunseich at NINDS. Hereditary spastic paraplegia; HSP, also known as familial spastic paraparesis, refers to a group of inherited disorders that involves weakness and spasticity. HSP is caused by autosomal recessive mutations in the genes encoding proteins forming AP-4 adaptor complex. Skin fibroblasts from a patient with biallelic variants in AP4M1 gene from Dr. Carsten Bonnemann, NICHD, were reprogrammed to hiPSC lines using Sendai virus. Epilepsy: Epilepsy is a group of neurological disorders characterized by recurrent seizures or disturbances in the electrical activity of the brain. Epilepsy affects people of all ages, from infants to the aged. Epilepsy can result from genetic variations, illness, head injury, or abnormal brain development. To generate cellular model for epilepsy, following epileptic genes were disrupted using CRISPR/cas9 genome modification technology; DNM1 which encodes a member of the dynamin superfamily of GTP-binding proteins; FRRS1L which encodes a component of the outer core of an AMPA receptor protein in the brain; CERS1 which encodes a ceramide synthase enzyme which catalyzed the synthesis of ceramide; CNTN2 which encodes a member of the contactin family of protein; GABRB3 which encodes a member of the ligand-gated ionic channel family. p53 mutation: In collaboration with Dr. Curtis Harris, NCI, we are stydtubg a unique p53 mutation in H9 hESCs, which was initially identified by Dr. Harris lab. This mutation appears to influence genomic stability and hPSC differentiation trajectories. 3. Reporter cell line generation GLIA markers: We have been developing reliable protocols to differentiate hiPSCs into glial cells. As part of this, three endogenous glia-specific proteins GFAP, OLIG2, and CX3CR1, have been tagged with GFP. The expression of GFP is being assessed after proper differentiation. Solute carrier proteins: The solute carrier protein (SLC) group of membrane transport proteins include over 400 members organized into 66 families. Most members of the SLC group are located in the cell membrane. Most SLC members function as transporters. Among SLC members, two neuron-specific glutamate transporters were tagged with GFP using CRISPR/cas9-assisted genome manipulation; SLC1A6 which encodes high-affinity glutamate transmembrane transporter involved in neurotransmitter uptake; SLC17A7 which encodes a glutamate transporter associated with the membranes of synaptic vesicles. Train scientists and technicians and provide technical support at the NIH: We have provided comprehensive training to six scientists or trainees at the NIH this year including the culture and cellular differentiation of hiPSCs, the formulation of differentiation protocols based on developmental principles and characterization of differentiated cells.