Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells.

BACKGROUND: Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. METHODS: In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single-cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells. RESULTS: We found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage. CONCLUSIONS: To our knowledge, this work established the first 3D hydrogel system for in situ differentiation of human iPSCs into PPs.

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.

Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution.

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.

Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells.

Background Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. Methods In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells. Results We found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage. Conclusions To our knowledge, this work established the first 3D hydrogel system for in situ differentiation of human iPSCs into PPs. Ongoing work focuses on enhancing pancreatic differentiation efficiency through modulating physicochemical properties of the iPSC-laden matrices.

CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids.

Mutations in CHD7 cause CHARGE syndrome, affecting multiple organs including the inner ear in humans. We investigate how CHD7 mutations affect inner ear development using human pluripotent stem cell-derived organoids as a model system. We find that loss of CHD7 or its chromatin remodeling activity leads to complete absence of hair cells and supporting cells, which can be explained by dysregulation of key otic development-associated genes in mutant otic progenitors. Further analysis of the mutant otic progenitors suggests that CHD7 can regulate otic genes through a chromatin remodeling-independent mechanism. Results from transcriptome profiling of hair cells reveal disruption of deafness gene expression as a potential underlying mechanism of CHARGE-associated sensorineural hearing loss. Notably, co-differentiating CHD7 knockout and wild-type cells in chimeric organoids partially rescues mutant phenotypes by restoring otherwise severely dysregulated otic genes. Taken together, our results suggest that CHD7 plays a critical role in regulating human otic lineage specification and hair cell differentiation.

Directed Differentiation of Human Pluripotent Stem Cells into Inner Ear Organoids.

The sensory epithelia of the inner ear contain mechanosensitive hair cells that detect sound and head acceleration. This protocol details a 3D differentiation method to generate inner ear organoids containing sensory epithelia with hair cells. Human pluripotent stem cells are aggregated in low-binding 96-well plates and treated in chemically defined media with extracellular matrix to promote epithelialization. Small molecules and recombinant proteins are applied in a stepwise manner to recapitulate the morphogenic cues (BMP, TGF-ß, FGF, and WNT) present during inner ear development in vivo. These treatments induce the sequential formation of nonneural ectoderm, otic-epibranchial progenitor domain, and otic placodes. The derived otic placodes then undergo self-guided morphogenesis to form otic vesicles, which eventually give rise to sensory epithelia containing hair cells and supporting cells, as well as neurons with synaptic formations to hair cells. This human stem cell-derived inner ear organoid system provides an ideal platform to study human inner ear development and disease in vitro.

Social Defeat Stress in Adolescent Mice Induces Depressive-like Behaviors with Reduced Oligodendrogenesis.

Strong stress related to adverse experiences during adolescence can cause mental disorders, as well as affecting brain structure and function. However, the underlying neurobiological mechanisms remain largely unknown. To investigate whether stress induced by adverse experience during adolescence affects oligodendrocyte (OL) remodeling, social defeat stress was applied to 6-week-old adolescent mice for 10 days, followed by behavioral tests and assessments of oligodendrogenesis. Socially defeated mice showed depressive-like behaviors in behavioral experiments. Stress led to a decrease in the number of newly born OLs in the anterior cortical region and the number of proteolipid protein-positive mature OLs in the corpus callosum and posterior cerebral cortex. Fewer bromodeoxyuridine-incorporated CC1-positive mature OLs were observed in these regions in socially defeated mice. To assess whether decreased oligodendrogenesis caused by social defeat stress is related to depressive-like symptoms under stress, clemastine, a drug that induces OL generation, was administered to socially defeated adolescent mice, resulting in the rescue of the behavioral abnormalities accompanied by increased oligodendrogenesis. These findings suggest that oligodendrogenesis in adverse environments during adolescence plays a role in psychiatric disorders, and clemastine may provide a potential therapeutic drug for adolescent mental disorders, targeting OLs.

Dynamic Interaction between Cortico-Brainstem Pathways during Training-Induced Recovery in Stroke Model Rats.

Reorganization of residual descending motor circuits underlies poststroke recovery. We previously clarified a causal relationship between the cortico-rubral tract and intensive limb use-induced functional recovery after internal capsule hemorrhage (ICH). However, other descending tracts, such as the cortico-reticular tract, might also be involved in rehabilitation-induced compensation. To investigate whether rehabilitation-induced recovery after ICH involves a shift in the compensatory circuit from the cortico-rubral tract to the cortico-reticular tract, we established loss of function of the cortico-rubral tract or/and cortico-reticular tract using two sets of viral vectors comprising the Tet-on system and designer receptors exclusively activated by the designer drug system. We used an ICH model that destroyed almost 60% of the corticofugal fibers. Anterograde tracing in rehabilitated rats revealed abundant sprouting of axons from the motor cortex in the red nucleus but not in the medullary reticular formation during the early phase of recovery. This primary contribution of the cortico-rubral tract was demonstrated by its selective blockade, whereas selective cortico-reticular tract silencing had little effect. Interestingly, cortico-rubral tract blockade from the start of rehabilitation induced an obvious increase of axon sprouting in the reticular formation with substantial functional recovery. Additional cortico-reticular tract silencing under the cortico-rubral tract blockade significantly worsened the recovered forelimb function. Furthermore, the alternative recruitment of the cortico-reticular tract was gradually induced by intensive limb use under cortico-rubral tract blockade, in which cortico-reticular tract silencing caused an apparent motor deficit. These findings indicate that individual cortico-brainstem pathways have dynamic compensatory potency to support rehabilitative functional recovery after ICH.SIGNIFICANCE STATEMENT This study aimed to clarify the interaction between the cortico-rubral and the cortico-reticular tract during intensive rehabilitation and functional recovery after capsular stroke. Pathway-selective disturbance by two sets of viral vectors revealed that the cortico-rubral tract was involved in rehabilitation-induced recovery of forelimb function from an early phase after internal capsule hemorrhage, but that the cortico-reticular tract was not. The sequential disturbance of both tracts revealed that the cortico-reticular tract was recruited and involved in rehabilitation-induced recovery when the cortico-rubral tract failed to function. Our data demonstrate a dynamic compensatory action of individual cortico-brainstem pathways for recovery through poststroke rehabilitation.

Alterations of Both Dendrite Morphology and Weaker Electrical Responsiveness in the Cortex of Hip Area Occur Before Rearrangement of the Motor Map in Neonatal White Matter Injury Model.

Hypoxia-ischemia (H-I) in rats at postnatal day 3 causes disorganization of oligodendrocyte development in layers II/III of the sensorimotor cortex without apparent neuronal loss, and shows mild hindlimb dysfunction with imbalanced motor coordination. However, the mechanisms by which mild motor dysfunction is induced without loss of cortical neurons are currently unclear. To reveal the mechanisms underlying mild motor dysfunction in neonatal H-I model, electrical responsiveness and dendrite morphology in the sensorimotor cortex were investigated at 10 weeks of age. Responses to intracortical microstimulation (ICMS) revealed that the cortical motor map was significantly changed in this model. The cortical area related to hip joint movement was reduced, and the area related to trunk movement was increased. Sholl analysis in Golgi staining revealed that layer I-III neurons on the H-I side had more dendrite branches compared with the contralateral side. To investigate whether changes in the motor map and morphology appeared at earlier stages, ICMS and Sholl analysis were also performed at 5 weeks of age. The minimal ICMS current to evoke twitches of the hip area was higher on the H-I side, while the motor map was unchanged. Golgi staining revealed more dendrite branches in layer I-III neurons on the H-I side. These results revealed that alterations of both dendrite morphology and ICMS threshold of the hip area occurred before the rearrangement of the motor map in the neonatal H-I model. They also suggest that altered dendritic morphology and altered ICMS responsiveness may be related to mild motor dysfunction in this model.

Monosodium glutamate ingestion during the development period reduces aggression mediated by the vagus nerve in a rat model of attention deficit-hyperactivity disorder.

We used an umami substance, monosodium glutamate (MSG), as a simple stimulant to clarify the mechanism of the formation of emotional behavior. A 60 mM MSG solution was fed to spontaneously hypertensive rats (SHR), used as a model of attention-deficit hyperactivity disorder, from postnatal day 25 for 5 weeks kept in isolation. Emotional behaviors (anxiety and aggression) were then assessed by the open-field test, cylinder test and social interaction test. MSG ingestion during the developmental period resulted in a significant reduction in aggressive behavior but had few effects on anxiety-like behavior. Several experiments were performed to identify the reason for the reduced aggression with MSG intake. Blood pressure in the MSG-treated SHR was comparable to that of the controls during development. Argyrophil III staining to detect the very early phase of neuronal damage revealed no evidence of injury by MSG in aggression-related brain areas. Assessment of plasma amino acids revealed that glutamate levels remained constant (∼80 µM) with MSG ingestion, except for a transient increase after fasting (∼700 µM). However, lactate dehydrogenase assay in an in vitro blood-brain barrier model showed that cell toxicity was not induced by indirect MSG application even at 700 µM, confirming that MSG ingestion caused minimal neuronal damage. Finally, vagotomy at the sub-diaphragmatic level before MSG ingestion blocked its effect on aggressive behavior in the isolated SHR. The data suggest that MSG ingestion during the developmental period can reduce aggressive behavior in an attention deficit-hyperactivity disorder model rat, mediated by gut-brain interaction.

Disorganization of Oligodendrocyte Development in the Layer II/III of the Sensorimotor Cortex Causes Motor Coordination Dysfunction in a Model of White Matter Injury in Neonatal Rats.

We previously established neonatal white matter injury (WMI) model rat that is made by right common carotid artery dissection at postnatal day 3, followed by 6% hypoxia for 60 min. This model has fewer oligodendrocyte progenitor cells and reduced myelin basic protein (MBP) positive areas in the sensorimotor cortex, but shows no apparent neuronal loss. However, how motor deficits are induced in this model is unclear. To elucidate the relationship between myelination disturbance and concomitant motor deficits, we first performed motor function tests (gait analysis, grip test, horizontal ladder test) and then analyzed myelination patterns in the sensorimotor cortex using transmission electron microscopy (TEM) and Contactin associated protein 1 (Caspr) staining in the neonatal WMI rats in adulthood. Behavioral tests revealed imbalanced motor coordination in this model. Motor deficit scores were higher in the neonatal WMI model, while hindlimb ladder stepping scores and forelimb grasping force were comparable to controls. Prolonged forelimb swing times and decreased hindlimb paw angles on the injured side were revealed by gait analysis. TEM revealed no change in myelinated axon number and the area g-ratio in the layer II/III of the cortex. Electromyographical durations and latencies in the gluteus maximus in response to electrical stimulation of the brain area were unchanged in the model. Caspr staining revealed fewer positive dots in layers II/III of the WMI cortex, indicating fewer and/or longer myelin sheath. These data suggest that disorganization of oligodendrocyte development in layers II/III of the sensorimotor cortex relates to imbalanced motor coordination in the neonatal WMI model rat.

A novel biosensor with high signal-to-noise ratio for real-time measurement of dopamine levels in vivo.

Fast-scan cyclic voltammetry (FSCV) is an established method for measuring dopamine (DA) levels in the brain in real time. However, it is difficult to discriminate DA from other monoamines such as serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE). We report a novel DA-specific biosensor consisting of a carbon-fiber electrode coated with an ion-exchange membrane, a layer containing monoamine oxidase B, and a cellulose membrane. We performed FSCV using the probe to monitor the amount of DA in vitro and in vivo. First, we measured currents in vitro in phosphate-buffered saline as we added one micromole each of DA, 5-HT, and NE. The results confirmed that the biosensor selectively detected DA. Next, we implanted the probe in the striatum of male rats to investigate whether it could selectively detect changes in the DA content in vivo. The probe detected both the tonic change induced by methamphetamine administration and the phasic change induced by electrical stimulation of the medial forebrain bundle. In contrast, the electrode in the 6-hydroxydopamine-lesioned striatum did not respond to systemic selective serotonin or serotonin/norepinephrine reuptake inhibitors, confirming its selectivity. Furthermore, the probe in the striatum could still detect changes in the DA level 1 week after electrode implantation. The results suggest that the novel biosensor can measure real-time changes in DA levels in vivo with a relatively high signal-to-noise ratio.

Dysfunction in Motor Coordination in Neonatal White Matter Injury Model Without Apparent Neuron Loss.

We made a white matter injury (WMI) model with mild hindlimb dysfunction by right common carotid artery occlusion followed by 6% oxygen for 60 min at postnatal day 3 (P3), in which actively proliferating oligodendrocyte (OL) progenitors are mainly damaged. To know whether this model is appropriate for cell therapy using OL progenitors, the pathological response to mild hypoxia-ischemia (H-I) in neurons and OL lineage cells and myelination failure were investigated along with gene expression analysis. In WMI model rats, coordinated motor function, as assessed by the accelerating rotarod test, was impaired. The dysfunction was accompanied by myelination failure in layers I-IV of the sensorimotor cortex. Although several oligo2-positive OLs stained positive for active caspase 3 in the cortex and white matter at 24 h after H-I, few NeuN-positive neurons were apoptotic. Argyrophil-III staining for damaged neurons revealed no increase in the number of degenerating cells in the model. Moreover, the total number of NeuN-positive neurons in the cortex was comparable to that of controls 7 days later. Retrograde labeling of the corticospinal tract with Fluoro-Gold revealed no significant loss of layer V neurons. In addition, no decrease in the numbers of cortical projecting neurons and layers V-VI neurons in both motor and sensory areas was observed. Interestingly, the numbers of inhibitory GABAergic cells immunoreactive for parvalbumin, calretinin, or somatostatin were preserved in the P26 cortex. Gene expression analysis at P5 revealed 98 upregulated and 65 downregulated genes that may relate to cell survival, myelin loss, and differentiation of OLs. These data suggest that impaired motor coordination was not induced by neuron loss but, rather, myelination failure in layers I-IV. As OL lineage cells are mainly damaged, this WMI model might be useful for cell-based therapy by replacing OL progenitors.

Early constraint-induced movement therapy promotes functional recovery and neuronal plasticity in a subcortical hemorrhage model rat.

Constraint-induced movement therapy (CIMT) promotes functional recovery of impaired forelimbs after hemiplegic strokes, including intracerebral hemorrhage (ICH). We used a rat model of subcortical hemorrhage to compare the effects of delivering early or late CIMT after ICH. The rat model was made by injecting collagenase into the globus pallidus near the internal capsule, and then forcing rats to use the affected forelimb for 7 days starting either 1 day (early CIMT) or 17 days (late CIMT) after the lesion. Recovery of forelimb function in the skilled reaching test and the ladder stepping test was found after early-CIMT, while no significant recovery was shown after late CIMT or in the non-CIMT controls. Early CIMT was associated with greater numbers of ΔFosB-positive cells in the ipsi-lesional sensorimotor cortex layers II-III and V. Additionally, we found expression of the growth-related genes brain-derived neurotrophic factor (BDNF) and growth-related protein 43 (GAP-43), and abundant dendritic arborization of pyramidal neurons in the sensorimotor area. Similar results were not detected in the contra-lesional cortex. In contrast to early CIMT, late CIMT failed to induce any changes in plasticity. We conclude that CIMT induces molecular and morphological plasticity in the ipsi-lesional sensorimotor cortex and facilitates better functional recovery when initiated immediately after hemorrhage.

Enhanced electrical responsiveness in the cerebral cortex with oral melatonin administration after a small hemorrhage near the internal capsule in rats.

Intracerebral hemorrhage (ICH) can cause direct brain injury at the insult site and indirect damage in remote brain areas. Although a protective effect of melatonin (ML) has been reported for ICH, its detailed mechanisms and effects on remote brain injury remain unclear. To clarify the mechanism of indirect neuroprotection after ICH, we first investigated whether ML improved motor function after ICH and then examined the underlying mechanisms. The ICH model rat was made by collagenase injection into the left globus pallidus, adjacent to the internal capsule. ML oral administration (15 mg/kg) for 7 days after ICH resulted in significant recovery of motor function. Retrograde labeling of the corticospinal tract by Fluoro-Gold revealed a significant increase in numbers of positive neurons in the cerebral cortex. Immunohistological analysis showed that ML treatment induced no difference in OX41-positive activated microglia/macrophage at day 1 (D1) but a significant reduction in 8-hydroxydeoxyguanosin-positive cells at D7. Neutral red assay revealed that ML significantly prevented H2 O2 -induced cell death in cultured oligodendrocytes and astrocytes but not in neurons. Electrophysiological response in the cerebral cortex area where the number of Fluoro-Gold-positive cells was increased was significantly improved in ML-treated rats. These data suggest that ML improves motor abilities after ICH by protecting oligodendrocytes and astrocytes in the vicinity of the lesion in the corticospinal tract from oxidative stress and causes enhanced electrical responsiveness in the cerebral cortex remote to the ICH pathology.

Differentiation of oligodendrocytes from mouse induced pluripotent stem cells without serum.

Cell therapy using induced pluripotent stem (iPS) cells might become a new approach for treating neonatal hypoxic-ischemic injury such as periventricular leukomalacia. To obtain appropriate donor cells for transplantation, we differentiated oligodendrocyte (OL) lineage cells from mouse iPS cells. Induction of OL lineage cell differentiation from iPS cells was carried out with a seven-step culture method. Mouse iPS cells (stage 1) were induced to form embryoid bodies for 4 days under a serum-free condition that was suitable for ectoderm induction (stage 2), following by selection of nestin-positive neural stem cells (NSCs) for 10-12 days (stage 3). NSCs were cultured in expansion medium containing fibroblast growth factor (FGF)-2 for 4 days (stage 4), induced to differentiate into glial progenitor cells by epidermal growth factor and fibroblast growth factor (FGF-2) treatment for 4-5 days (stage 5), and then into OL progenitor cells by culture in neurobasal A medium containing FGF-2 and platelet-derived growth factor for 6-8 days (stage 6). Terminal differentiation into O4-positive OLs was carried out by culture in neurobasal A containing T3 and ciliary neurotrophic factor for 7 days (stage 7). Inwardly rectifying K+ currents, which are characteristic of OLs, were detected in iPS cell-derived cells at stage 7 in whole cell clamp mode. Our data suggest that OLs can be effectively differentiated from mouse iPS cells without serum in a stepwise manner, which may be appropriate for use as donor cells in transplantation.

Minor neuronal damage and recovered cellular proliferation in the hippocampus after continuous unilateral forelimb restraint in normal rats.

Constraint-induced movement therapy (CIMT) involves the restraint of an intact limb to force the dominant use of an affected limb, in an attempt to enhance use-dependent plasticity and reduce dysfunction. To investigate whether forced disuse of an intact forelimb with CIMT causes a loss of limb function and degenerative damage in the brain, a staircase test and a horizontal ladder test were carried out in control rats and forelimb-restrained rats, and then Argyrophil III silver staining, which is capable of detecting subtle neuronal damage, was used to examine histological alterations associated with restraint. No significant changes in forelimb function were observed in restrained rats. However, atypical weak argyrophilic neurons, an indicator of minor neural damage, were found in the bilateral hippocampus of restrained rats. This damage was not found in the cortex, striatum, or spinal cord. Investigation of neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ) revealed a clear reduction in the number of bromodeoxyuridine-positive cells in bilateral SGZ, but not in the SVZ, in restrained rats compared with controls. This reduction was accompanied by reduced mRNA expression of vascular endothelial growth factor and glial-derived neurotrophic factor. However, reduced cellular proliferation and decreased gene expression were recovered after the removal of the restraint. Our results suggest that forced disuse of the intact forelimb has no significant effect on skilled forelimb function but has a minor effect on neurogenesis in SGZ, suggesting that mild stress may be caused by the restraint.