Functions and interactions of homeodomain proteins during development of the spinal cord

Das dorsale Rückenmark besteht aus verschiedenen Populationen von Interneuronen, die sensorische Informationen aus der Peripherie verarbeiten und weiterleiten. Diese dorsalen Interneurone entstehen in zwei zeitlich versetzten Phasen. Sechs Populationen von "frühen" dorsalen Interneuronen (dI1-dI6) entstehen während der ersten Phase, wohingegen zwei unterschiedliche Populationen von "späten" Interneuronen (dILA/B) während der zweiten Phase der Neurogenese gebildet werden. Diese Arbeit untersucht die Rolle zweier Homöodomänenproteine, (Gsh1 und Gsh2, bei der Spezifizierung von Interneuronen in der dorsalen Hälfte des Rückenmarks. Gsh1 und Gsh2 werden in drei verschiedenen Vorläuferdomänen von frühen (dI3-5) Interneuronen, als auch in der Vorläuferdomäne der zwei späten dorsalen Interneuronen exprimiert. Während der ersten Phase der Neurogenese sind Gsh1 und Gsh2 notwendig für die Entwicklung der erregenden dI3 und dI5 Neurone. Sie erhalten hierbei die Expression des bHLH (basic Helix-Loop-Helix) Proteins Mash1, ein essentieller Faktor für ein erregendes dI3 und dI5 Zellschicksal, aufrecht, indem sie den bHLH Transkriptionsfaktor Ngn1 hemmen. Die Expression dieses Proteins grenzt im Wildtyp sowohl dorsal als auch ventral an die Gsh1/2+ Domäne an. Ngn1 expandiert im Rückenmark von Gsh1/2 Doppel-Mutanten, was zu einer starken Reduktion von Mash1 führt. Diese Reduktion wiederum resultiert im Fehlen der dI3 und dI5 Populationen von dorsalen Interneuronen. Gsh1 und Gsh2 sind außerdem notwendig für die Spezifizierung der erregenden dILB Neurone, denn diese Population der späten Interneurone fehlt im Rückenmark von Gsh1/2-defizienten Mausembryonen. Interessanterweise wechselt Mash1 seine Funktion während der zweiten Phase der Neurogenese im dorsalen Rückenmark. Während dieser Phase arbeitet Mash1 gegen Gsh1/2 und ist nun notwendig für die Spezifizierung inhibierender dILA Neurone indem es die Expression von Ptf1a in den Vorläufern der dILA Neurone induziert. Dieser bHLH Transkriptionsfaktor hemmt die Funktion von Gsh1/2 und begünstigt dadurch die Entwicklung von inhibierenden dILA Neuronen aus Gsh1/2 positiven Vorläufern. Zusammenfassend identifiziert diese Arbeit die Homöodomänenproteine Gsh1/2 und den bHLH Transkriptionsfaktor Mash1 als Hauptfaktoren bei der Etablierung eines erregenden, bzw. inhibierenden Zellschicksals während der Entwicklung des dorsalen Rückenmarks

Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE

Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurrl(+) stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery

The Polycomb Group Protein L3MBTL1 Represses a SMAD5-Mediated Hematopoietic Transcriptional Program in Human Pluripotent Stem Cells

SummaryEpigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia

Gbx1 and Gbx2 Are Essential for Normal Patterning and Development of Interneurons and Motor Neurons in the Embryonic Spinal Cord

The molecular mechanisms regulating neurogenesis involve the control of gene expression by transcription factors. Gbx1 and Gbx2, two members of the Gbx family of homeodomain-containing transcription factors, are known for their essential roles in central nervous system development. The expression domains of mouse Gbx1 and Gbx2 include regions of the forebrain, anterior hindbrain, and spinal cord. In the spinal cord, Gbx1 and Gbx2 are expressed in PAX2+ interneurons of the dorsal horn and ventral motor neuron progenitors. Based on their shared domains of expression and instances of overlap, we investigated the functional relationship between Gbx family members in the developing spinal cord using Gbx1−/−, Gbx2−/−, and Gbx1−/−/Gbx2−/− embryos. In situ hybridization analyses of embryonic spinal cords show upregulation of Gbx2 expression in Gbx1−/− embryos and upregulation of Gbx1 expression in Gbx2−/− embryos. Additionally, our data demonstrate that Gbx genes regulate development of a subset of PAX2+ dorsal inhibitory interneurons. While we observe no difference in overall proliferative status of the developing ependymal layer, expansion of proliferative cells into the anatomically defined mantle zone occurs in Gbx mutants. Lastly, our data shows a marked increase in apoptotic cell death in the ventral spinal cord of Gbx mutants during mid-embryonic stages. While our studies reveal that both members of the Gbx gene family are involved in development of subsets of PAX2+ dorsal interneurons and survival of ventral motor neurons, Gbx1 and Gbx2 are not sufficient to genetically compensate for the loss of one another. Thus, our studies provide novel insight to the relationship harbored between Gbx1 and Gbx2 in spinal cord development

Specification of Functional Cranial Placode Derivatives from Human Pluripotent Stem Cells

Cranial placodes are embryonic structures essential for sensory and endocrine organ development. Human placode development has remained largely inaccessible despite the serious medical conditions caused by the dysfunction of placode-derived tissues. Here, we demonstrate the efficient derivation of cranial placodes from human pluripotent stem cells. Timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Concomitant inhibition of fibroblast growth factor signaling disrupts placode derivation and induces surface ectoderm. Further fate specification at the preplacode stage enables the selective generation of placode-derived trigeminal ganglia capable of in vivo engraftment, mature lens fibers, and anterior pituitary hormone-producing cells that upon transplantation produce human growth hormone and adrenocorticotropic hormone in vivo. Our results establish a powerful experimental platform to study human cranial placode development and set the stage for the development of human cell-based therapies in sensory and endocrine disease

A Cell Engineering Strategy to Enhance the Safety of Stem Cell Therapies

The long-term risk of malignancy associated with stem cell therapies is a significant concern in the clinical application of this exciting technology. We report a cancer-selective strategy to enhance the safety of stem cell therapies. Briefly, using a cell engineering approach, we show that aggressive cancers derived from human or murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are strikingly sensitive to temporary MYC blockade. On the other hand, differentiated tissues derived from human or mouse iPSCs can readily tolerate temporary MYC inactivation. In cancer cells, endogenous MYC is required to maintain the metabolic and epigenetic functions of the embryonic and cancer-specific pyruvate kinase M2 isoform (PKM2). In summary, our results implicate PKM2 in cancer’s increased MYC dependence and indicate dominant MYC inhibition as a cancer-selective fail-safe for stem cell therapies

Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE

Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial