Huisheng Liu

Human Embryonic Stem Cell-Derived GABA Neurons Correct Locomotion Deficits in Quinolinic Acid-Lesioned Mice

Degeneration of medium spiny GABA neurons in the basal ganglia underlies motor dysfunction in Huntingtons disease (HD), which presently lacks effective therapy. In this study, we have successfully directed human embryonic stem cells (hESCs) to enriched populations of DARPP32-expressing forebrain GABA neurons. Transplantation of these human forebrain GABA neurons and their progenitors, but not spinal GABA cells, into the striatum of quinolinic acid-lesioned mice results in generation of large populations of DARPP32(+) GABA neurons, which project to the substantia nigra as well as receiving glutamatergic and dopaminergic inputs, corresponding to correction of motor deficits. This finding raises hopes for cell therapy for HD.

Modeling ALS with iPSCs Reveals that Mutant SOD1 Misregulates Neurofilament Balance in Motor Neurons

Amyotrophic lateral sclerosis (ALS) presents motoneuron (MN)-selective protein inclusions and axonal degeneration but the underlying mechanisms of such are unknown. Using induced pluripotent cells (iPSCs) from patients with mutation in the Cu/Zn superoxide dismutase (SOD1) gene, we show that spinal MNs, but rarely non-MNs, exhibited neurofilament (NF) aggregation followed by neurite degeneration when glia were not present. These changes were associated with decreased stability of NF-L mRNA and binding of its 3 UTR by mutant SOD1 and thus altered protein proportion of NF subunits. Such MN-selective changes were mimicked by expression of a single copy of the mutant SOD1 in human embryonic stem cells and were prevented by genetic correction of the SOD1 mutation in patients iPSCs. Importantly, conditional expression of NF-L in the SOD1 iPSC-derived MNs corrected the NF subunit proportion, mitigating NF aggregation and neurite degeneration. Thus, NF misregulation underlies mutant SOD1-mediated NF aggregation and axonal degeneration in ALS MNs.

Medial ganglionic eminence–like cells derived from human embryonic stem cells correct learning and memory deficits

Dysfunction of basal forebrain cholinergic neurons (BFCNs) and γ-aminobutyric acid (GABA) interneurons, derived from medial ganglionic eminence (MGE), is implicated in disorders of learning and memory. Here we present a method for differentiating human embryonic stem cells (hESCs) to a nearly uniform population of NKX2.1+ MGE-like progenitor cells. After transplantation into the hippocampus of mice in which BFCNs and some GABA neurons in the medial septum had been destroyed by mu P75-saporin, human MGE-like progenitors, but not ventral spinal progenitors, produced BFCNs that synaptically connected with endogenous neurons, whereas both progenitors generated similar populations of GABA neurons. Mice transplanted with MGE-like but not spinal progenitors showed improvements in learning and memory deficits. These results suggest that progeny of the MGE-like progenitors, particularly BFCNs, contributed to learning and memory. Our findings support the prospect of using human stem cell–derived MGE-like progenitors in developing therapies for neurological disorders of learning and memory.

Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells

Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA interneuron deficit disease, will be instrumental to the investigation of disease pathogenesis and development of therapeutics. We describe a protocol for directed differentiation of forebrain GABA interneurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a chemically defined system. In this protocol, human PSCs are first induced to primitive neuroepithelial cells over 10 d, and then patterned to NKX2.1-expressing medial ganglionic eminence progenitors by simple treatment with sonic hedgehog or its agonist purmorphamine over the next 2 weeks. These progenitors generate a nearly pure population of forebrain GABA interneurons by the sixth week. This simple and efficient protocol does not require transgenic modification or cell sorting, and it has been replicated with multiple human ESC and iPSC lines.

Specification of Midbrain Dopamine Neurons from Primate Pluripotent Stem Cells

By sequentially applying sonic hedgehog (C25II) and CHIR99021 (GSK3β inhibitor) to induce the midbrain floor plate (FP) progenitors and fibroblast growth factor 8 (FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for the generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells and induced pluripotent stem cells (PSCs). We found that CHIR99021 specifies diencephalon to hind brain fates in a concentration‐dependent manner and only a narrow concentration range of CHIR99021 at a particular window is necessary to induce the midbrain FP progenitors, expressing Corin, En1, FoxA2, and Lmx1a. FGF8 enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of tyrosine hydroxylase, Lmx1a/b, FoxA2, FoxP1, Nurr1, and En1 as well as typical electrophysiological properties. More than half of these DA neurons expressed A9 DA neuron markers Girk2 and ALDH1a1. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy. STEM CELLS2012;30:1655–1663

Generation of integration-free and region-specific neural progenitors from primate fibroblasts.

Postnatal and adult human and monkey fibroblasts were infected with Sendai virus containing the Yamanaka factors for 24xa0hr, then they were cultured in a chemically defined medium containing leukemia inhibitory factor (LIF), transforming growth factor (TGF)-β inhibitor SB431542, and glycogen synthase kinase (GSK)-3β inhibitor CHIR99021 at 39°C for inactivation of the virus. Induced neural progenitor (iNP) colonies appeared as early as day 13 and can be expanded for >20 passages. Under the same defined condition, no induced pluripotent stem cell (iPSC) colonies formed at either 37°C or 39°C. The iNPs predominantly express hindbrain genes and differentiate into hindbrain neurons, and when caudalized, they produced an enriched population of spinal motor neurons. Following transplantation into the forebrain, the iNP-derived cells retained the hindbrain identity. The ability to generate defined, integration-free iNPs from adult primate fibroblasts under a defined condition with predictable fate choices will facilitate disease modeling and therapeutic development.

Generation and Expansion of highly-pure Motor Neuron Progenitors from Human Pluripotent Stem Cells

SUMMARY Human 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.

Generation of serotonin neurons from human pluripotent stem cells

Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2-3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.

Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson’s Disease

Transplantation of human pluripotent stem cell (hPSC)-derived neurons is a promising avenue for treating disorders including Parkinsons disease (PD). Precise control over engrafted cell activity is highly desired, as cells do not always integrate properly into host circuitry and can cause suboptimal graft function or undesired outcomes. Here, we show tunable rescue of motor function in a mouse model of PD, following transplantation of human midbrain dopaminergic (mDA) neurons differentiated from hPSCs engineered to express DREADDs (designer receptors exclusively activated by designer drug). Administering clozapine-N-oxide (CNO) enabled precise DREADD-dependent stimulation or inhibition of engrafted neurons, revealing D1 receptor-dependent regulation of host neuronal circuitry by engrafted cells. Transplanted cells rescued motor defects, which could be reversed or enhanced by CNO-based control of graft function, and activating engrafted cells drives behavioral changes in transplanted mice. These results highlight the ability to exogenously and noninvasively control and refine therapeutic outcomes following cell transplantation.

Human-derived neural progenitors functionally replace astrocytes in adult mice

Astrocytes are integral components of the homeostatic neural network as well as active participants in pathogenesis of and recovery from nearly all neurological conditions. Evolutionarily, compared with lower vertebrates and nonhuman primates, humans have an increased astrocyte-to-neuron ratio; however, a lack of effective models has hindered the study of the complex roles of human astrocytes in intact adult animals. Here, we demonstrated that after transplantation into the cervical spinal cords of adult mice with severe combined immunodeficiency (SCID), human pluripotent stem cell-derived (PSC-derived) neural progenitors migrate a long distance and differentiate to astrocytes that nearly replace their mouse counterparts over a 9-month period. The human PSC-derived astrocytes formed networks through their processes, encircled endogenous neurons, and extended end feet that wrapped around blood vessels without altering locomotion behaviors, suggesting structural, and potentially functional, integration into the adult mouse spinal cord. Furthermore, in SCID mice transplanted with neural progenitors derived from induced PSCs from patients with ALS, astrocytes were generated and distributed to a similar degree as that seen in mice transplanted with healthy progenitors; however, these mice exhibited motor deficit, highlighting functional integration of the human-derived astrocytes. Together, these results indicate that this chimeric animal model has potential for further investigating the roles of human astrocytes in disease pathogenesis and repair.