Xue Jun Li

Specification of motoneurons from human embryonic stem cells

An understanding of how mammalian stem cells produce specific neuronal subtypes remains elusive. Here we show that human embryonic stem cells generated early neuroectodermal cells, which organized into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube–like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro–generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage.

Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage

Understanding neuroectoderm formation and subsequent diversification to functional neural subtypes remains elusive. We show here that human embryonic stem cells (hESCs) differentiate to primitive neuroectoderm after 8–10 days. At this stage, cells uniformly exhibit columnar morphology and express neural markers, including anterior but not posterior homeodomain proteins. The anterior identity of these cells develops regardless of morphogens present during initial neuroectoderm specification. This anterior phenotype can be maintained or transformed to a caudal fate with specific morphogens over the next week, when cells become definitive neuroepithelia, marked by neural tube‐like structures with distinct adhesion molecule expression, Sox1 expression, and a resistance to additional patterning signals. Thus, primitive neuroepithelia represents the earliest neural cells that possess the potential to differentiate to regionally specific neural progenitors. This finding offers insights into early human brain development and lays a foundation for generating neural cells with correct positional and transmitter profiles.

Directed Differentiation of Ventral Spinal Progenitors and Motor Neurons from Human Embryonic Stem Cells by Small Molecules

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons (∼50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7−), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.

Pax6 Is a Human Neuroectoderm Cell Fate Determinant

The transcriptional regulation of neuroectoderm (NE) specification is unknown. Here we show that Pax6 is uniformly expressed in early NE cells of human fetuses and those differentiated from human embryonic stem cells (hESCs). This is in contrast to the later expression of Pax6 in restricted mouse brain regions. Knockdown of Pax6 blocks NE specification from hESCs. Overexpression of either Pax6a or Pax6b, but not Pax6triangle upPD, triggers hESC differentiation. However, only Pax6a converts hESCs to NE. In contrast, neither loss nor gain of function of Pax6 affects mouse NE specification. Both Pax6a and Pax6b bind to pluripotent gene promoters but only Pax6a binds to NE genes during human NE specification. These findings indicate that Pax6 is a transcriptional determinant of the human NE and suggest that Pax6a and Pax6b coordinate with each other in determining the transition from pluripotency to the NE fate in human by differentially targeting pluripotent and NE genes.

Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells

The directed differentiation of forebrain neuronal types from human embryonic stem cells (hESCs) has not been achieved. Here, we show that hESCs differentiate to telencephalic progenitors with a predominantly dorsal identity in a chemically defined medium without known morphogens. This is attributed to endogenous Wnt signaling, which upregulates the truncated form of GLI3, a repressor of sonic hedgehog (SHH). A high concentration of SHH, or the inhibition of Wnt by dickkopf 1 (DKK1) together with a low concentration of SHH, almost completely converts the primitive dorsal precursors to ventral progenitors, which is partially achieved through both downregulation of the truncated GLI3 and upregulation of full-length GLI3 expression. These dorsal and ventral telencephalic progenitors differentiate to functional glutamatergic and GABAergic neurons, respectively. Thus, although hESCs generate dorsal telencephalic cells, as opposed to ventral progenitors in other vertebrates, in the absence of exogenous morphogens, human cells use a similar molecular mechanism to control the dorsal versus ventral fate. The coordination of Wnt and SHH signaling through GLI3 represents a novel mechanism that regulates ventral-dorsal patterning in the development of forebrain neuronal subtypes.

Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects

Human embryonic stem cells (hESCs) offer a platform to bridge what we have learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, we show that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors.

Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells.

Background Directed differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation. Methodology/Principal Findings We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons. Conclusions/Significance Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.

Melatonin protects against MPTP/MPP+-induced mitochondrial DNA oxidative damage in vivo and in vitro

Abstract:  The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) and 1‐methyl‐4‐phenylpyridine ion (MPP+) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8‐hydroxyguanine (8‐oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose‐dependently prevented MPTP‐induced DNA oxidative damage. In SH‐SY5Y cells, MPP+ (1 mm) increased the immunoreactivity of 8‐oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 μm) preincubation significantly attenuated MPP+‐induced mtDNA oxidative damage. Furthermore, MPP+ time‐dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Ψm) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP+ caused cell death in 49% of the control. However, melatonin prevented MPP+‐induced mtOFR generation and Ψm collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP+‐induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Ψm collapse.

Loss of Spastin Function Results in Disease-Specific Axonal Defects in Human Pluripotent Stem Cell-Based Models of Hereditary Spastic Paraplegia

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease‐specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule‐severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC‐derived neurons recapitulate disease‐specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin‐knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin‐knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated‐tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule‐destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin‐knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell‐based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP. Stem Cells 2014;32:414–423

Induced pluripotent stem (iPS) cells as in vitro models of human neurogenetic disorders

The recent discovery of genomic reprogramming of human somatic cells into induced pluripotent stem cells offers an innovative and relevant approach to the study of human genetic and neurogenetic diseases. By reprogramming somatic cells from patient samples, cell lines can be isolated that self-renew indefinitely and have the potential to develop into multiple different tissue lineages. Additionally, the rapid progress of research on human embryonic stem cells has led to the development of sophisticated in vitro differentiation protocols that closely mimic mammalian development. In particular, there have been significant advances in differentiating human pluripotent stem cells into defined neuronal types. Here, we summarize the experimental approaches employed in the rapidly evolving area of somatic cell reprogramming and the methodologies for differentiating human pluripotent cells into neurons. We also discuss how the availability of patient-specific fibroblasts offers a unique opportunity for studying and modeling the effects of specific gene defects on human neuronal development in vitro and for testing small molecules or other potential therapies for the relevant neurogenetic disorders.