Thalamic Foxp2 regulates output connectivity and sensory-motor impairments in a model of Huntington's Disease.

BACKGROUND: Huntington's Disease (HD) is a disorder that affects body movements. Altered glutamatergic innervation of the striatum is a major hallmark of the disease. Approximately 30% of those glutamatergic inputs come from thalamic nuclei. Foxp2 is a transcription factor involved in cell differentiation and reported low in patients with HD. However, the role of the Foxp2 in the thalamus in HD remains unexplored. METHODS: We used two different mouse models of HD, the R6/1 and the HdhQ111 mice, to demonstrate a consistent thalamic Foxp2 reduction in the context of HD. We used in vivo electrophysiological recordings, microdialysis in behaving mice and rabies virus-based monosynaptic tracing to study thalamo-striatal and thalamo-cortical synaptic connectivity in R6/1 mice. Micro-structural synaptic plasticity was also evaluated in the striatum and cortex of R6/1 mice. We over-expressed Foxp2 in the thalamus of R6/1 mice or reduced Foxp2 in the thalamus of wild type mice to evaluate its role in sensory and motor skills deficiencies, as well as thalamo-striatal and thalamo-cortical connectivity in such mouse models. RESULTS: Here, we demonstrate in a HD mouse model a clear and early thalamo-striatal aberrant connectivity associated with a reduction of thalamic Foxp2 levels. Recovering thalamic Foxp2 levels in the mouse rescued motor coordination and sensory skills concomitant with an amelioration of neuropathological features and with a repair of the structural and functional connectivity through a restoration of neurotransmitter release. In addition, reduction of thalamic Foxp2 levels in wild type mice induced HD-like phenotypes. CONCLUSIONS: In conclusion, we show that a novel identified thalamic Foxp2 dysregulation alters basal ganglia circuits implicated in the pathophysiology of HD.

Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior.

Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus-based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.

Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli.

Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.

Monocyte-Derived Macrophages Contribute to Spontaneous Long-Term Functional Recovery after Stroke in Mice.

Stroke is a leading cause of disability and currently lacks effective therapy enabling long-term functional recovery. Ischemic brain injury causes local inflammation, which involves both activated resident microglia and infiltrating immune cells, including monocytes. Monocyte-derived macrophages (MDMs) exhibit a high degree of functional plasticity. Here, we determined the role of MDMs in long-term spontaneous functional recovery after middle cerebral artery occlusion in mice. Analyses by flow cytometry and immunocytochemistry revealed that monocytes home to the stroke-injured hemisphere., and that infiltration peaks 3 d after stroke. At day 7, half of the infiltrating MDMs exhibited a bias toward a proinflammatory phenotype and the other half toward an anti-inflammatory phenotype, but during the subsequent 2 weeks, MDMs with an anti-inflammatory phenotype dominated. Blocking monocyte recruitment using the anti-CCR2 antibody MC-21 during the first week after stroke abolished long-term behavioral recovery, as determined in corridor and staircase tests, and drastically decreased tissue expression of anti-inflammatory genes, including TGFß, CD163, and Ym1. Our results show that spontaneously recruited monocytes to the injured brain early after the insult contribute to long-term functional recovery after stroke. SIGNIFICANCE STATEMENT: For decades, any involvement of circulating immune cells in CNS repair was completely denied. Only over the past few years has involvement of monocyte-derived macrophages (MDMs) in CNS repair received appreciation. We show here, for the first time, that MDMs recruited to the injured brain early after ischemic stroke contribute to long-term spontaneous functional recovery through inflammation-resolving activity. Our data raise the possibility that inadequate recruitment of MDMs to the brain after stroke underlies the incomplete functional recovery seen in patients and that boosting homing of MDMs with an anti-inflammatory bias to the injured brain tissue may be a new therapeutic approach to promote long-term improvement after stroke.

Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke.

BACKGROUND: Choroid plexus (CP) supports the entry of monocyte-derived macrophages (MDMs) to the central nervous system in animal models of traumatic brain injury, spinal cord injury, and Alzheimer's disease. Whether the CP is involved in the recruitment of MDMs to the injured brain after ischemic stroke is unknown. METHODS: Adult male C57BL/6 mice were subjected to focal cortical ischemia by permanent occlusion of the distal branch of the right middle cerebral artery. Choroid plexus tissues were collected and analyzed for Vcam1, Madcam1, Cx3cl1, Ccl2, Nt5e, and Ifnγ expression at different timepoints after stroke using qPCR. Changes of MDMs in CP and cerebrospinal fluid (CSF) at 1 day and 3 days after stroke were analyzed using flow cytometry. Infiltration of MDMs into CP and CSF were validated using ß-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. CD115+ monocytes were isolated using a magnetic cell separation system from bone marrow of Cx3cr1-GFP or wild-type C57BL/6 donor mice. The freshly isolated monocytes or M2-like MDMs primed in vitro with IL4 and IL13 were stereotaxically injected into the lateral ventricle of stroke-affected mice to trace for their migration into ischemic hemisphere or to assess their effect on post-stroke recovery using open field, corridor, and active avoidance behavioral tests. RESULTS: We found that CP responded to cortical stroke by upregulation of gene expression for several possible mediators of MDM trafficking and, concomitantly, MDMs increased in CP and cerebrospinal fluid (CSF). We then confirmed that MDMs infiltrated from blood into CP and CSF after the insult using ß-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. When MDMs were directly administered into CSF following stroke, they homed to the ischemic hemisphere. If they had been primed in vitro prior to their administration to become M2-like macrophages, they promoted post-stroke recovery of motor and cognitive function without influencing infarct volume. CONCLUSIONS: Our findings suggest the possibility that autologous transplantation of M2-like MDMs into CSF might be developed into a new strategy for promoting recovery also in patients with stroke.

Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery.

Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term self-renewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated cell-transplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our findings show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intracortical implantation in a rat stroke model.

Updated Toolbox for Assessing Neuronal Network Reconstruction after Cell Therapy.

Cell therapy has proven to be a promising treatment for a range of neurological disorders, including Parkinson Disease, drug-resistant epilepsy, and stroke, by restoring function after brain damage. Nevertheless, evaluating the true effectiveness of these therapeutic interventions requires a deep understanding of the functional integration of grafted cells into existing neural networks. This review explores a powerful arsenal of molecular techniques revolutionizing our ability to unveil functional integration of grafted cells within the host brain. From precise manipulation of neuronal activity to pinpoint the functional contribution of transplanted cells by using opto- and chemo-genetics, to real-time monitoring of neuronal dynamics shedding light on functional connectivity within the reconstructed circuits by using genetically encoded (calcium) indicators in vivo. Finally, structural reconstruction and mapping communication pathways between grafted and host neurons can be achieved by monosynaptic tracing with viral vectors. The cutting-edge toolbox presented here holds immense promise for elucidating the impact of cell therapy on neural circuitry and guiding the development of more effective treatments for neurological disorders.

Human-induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain.

Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells survive long-term, differentiate to functional neurons, and induce recovery in the stroke-injured brain are unclear. We have transplanted long-term self-renewing neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs, into the stroke-damaged mouse and rat striatum or cortex. Recovery of forepaw movements was observed already at 1 week after transplantation. Improvement was most likely not due to neuronal replacement but was associated with increased vascular endothelial growth factor levels, probably enhancing endogenous plasticity. Transplanted cells stopped proliferating, could survive without forming tumors for at least 4 months, and differentiated to morphologically mature neurons of different subtypes. Neurons in intrastriatal grafts sent axonal projections to the globus pallidus. Grafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons. Our study provides the first evidence that transplantation of human iPSC-derived cells is a safe and efficient approach to promote recovery after stroke and can be used to supply the injured brain with new neurons for replacement.

Long-term calcium imaging reveals functional development in hiPSC-derived cultures comparable to human but not rat primary cultures.

Models for human brain-oriented research are often established on primary cultures from rodents, which fails to recapitulate cellular specificity and molecular cues of the human brain. Here we investigated whether neuronal cultures derived from human induced pluripotent stem cells (hiPSCs) feature key advantages compared with rodent primary cultures. Using calcium fluorescence imaging, we tracked spontaneous neuronal activity in hiPSC-derived, human, and rat primary cultures and compared their dynamic and functional behavior as they matured. We observed that hiPSC-derived cultures progressively changed upon development, exhibiting gradually richer activity patterns and functional traits. By contrast, rat primary cultures were locked in the same dynamic state since activity onset. Human primary cultures exhibited features in between hiPSC-derived and rat primary cultures, although traits from the former predominated. Our study demonstrates that hiPSC-derived cultures are excellent models to investigate development in neuronal assemblies, a hallmark for applications that monitor alterations caused by damage or neurodegeneration.

Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells.

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge. Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture. Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of ß-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

First Organoid Intelligence (OI) workshop to form an OI community.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

Rich dynamics and functional organization on topographically designed neuronal networks in vitro.

Neuronal cultures are a prominent experimental tool to understand complex functional organization in neuronal assemblies. However, neurons grown on flat surfaces exhibit a strongly coherent bursting behavior with limited functionality. To approach the functional richness of naturally formed neuronal circuits, here we studied neuronal networks grown on polydimethylsiloxane (PDMS) topographical patterns shaped as either parallel tracks or square valleys. We followed the evolution of spontaneous activity in these cultures along 20 days in vitro using fluorescence calcium imaging. The networks were characterized by rich spatiotemporal activity patterns that comprised from small regions of the culture to its whole extent. Effective connectivity analysis revealed the emergence of spatially compact functional modules that were associated with both the underpinned topographical features and predominant spatiotemporal activity fronts. Our results show the capacity of spatial constraints to mold activity and functional organization, bringing new opportunities to comprehend the structure-function relationship in living neuronal circuits.

Neuronal Replacement in Stem Cell Therapy for Stroke: Filling the Gap.

Stem cell therapy using human skin-derived neural precursors holds much promise for the treatment of stroke patients. Two main mechanisms have been proposed to give rise to the improved recovery in animal models of stroke after transplantation of these cells. First, the so called by-stander effect, which could modulate the environment during early phases after brain tissue damage, resulting in moderate improvements in the outcome of the insult. Second, the neuronal replacement and functional integration of grafted cells into the impaired brain circuitry, which will result in optimum long-term structural and functional repair. Recently developed sophisticated research tools like optogenetic control of neuronal activity and rabies virus monosynaptic tracing, among others, have made it possible to provide solid evidence about the functional integration of grafted cells and its contribution to improved recovery in animal models of brain damage. Moreover, previous clinical trials in patients with Parkinson's Disease represent a proof of principle that stem cell-based neuronal replacement could work in humans. Our studies with in vivo and ex vivo transplantation of human skin-derived cells neurons in animal model of stroke and organotypic cultures of adult human cortex, respectively, also support the hypothesis that human somatic cells reprogrammed into neurons can get integrated in the human lesioned neuronal circuitry. In the present short review, we summarized our data and recent studies from other groups supporting the above hypothesis and opening new avenues for development of the future clinical applications.

Poly(ester amide) microspheres are efficient vehicles for long-term intracerebral growth factor delivery and improve functional recovery after stroke.

Growth factors promote plasticity in injured brain and improve impaired functions. For clinical application, efficient approaches for growth factor delivery into the brain are necessary. Poly(ester amide) (PEA)-derived microspheres (MS) could serve as vehicles due to their thermal and mechanical properties, biocompatibility and biodegradability. Vascular endothelial growth factor (VEGF) exerts both vascular and neuronal actions, making it suitable to stimulate post-stroke recovery. Here, PEA (composed of adipic acid, L-phenyl-alanine and 1,4-butanediol) MS were loaded with VEGF and injected intracerebrally in mice subjected to cortical stroke. Loaded MS provided sustained release of VEGF in vitro and, after injection, biologically active VEGF was released long-term, as evidenced by high VEGF immunoreactivity, increased VEGF tissue levels, and higher vessel density and more NG2+ cells in injured hemisphere of animals with VEGF-loaded as compared to non-loaded MS. Loaded MS gave rise to more rapid recovery of neurological score. Both loaded and non-loaded MS induced improvement in neurological score and adhesive removal test, probably due to anti-inflammatory action. In summary, grafted PEA MS can act as efficient vehicles, with anti-inflammatory action, for long-term delivery of growth factors into injured brain. Our data suggest PEA MS as a new tool for neurorestorative approaches with therapeutic potential.

Spontaneous Functional Recovery after Focal Damage in Neuronal Cultures.

Damage in biological neuronal networks triggers a complex functional reorganization whose mechanisms are still poorly understood. To delineate this reorganization process, here we investigate the functional alterations of in vitro rat cortical circuits following localized laser ablation. The analysis of the functional network configuration before and after ablation allowed us to quantify the extent of functional alterations and the characteristic spatial and temporal scales along recovery. We observed that damage precipitated a fast rerouting of information flow that restored network's communicability in about 15 min. Functional restoration was led by the immediate neighbors around trauma but was orchestrated by the entire network. Our in vitro setup exposes the ability of neuronal circuits to articulate fast responses to acute damage, and may serve as a proxy to devise recovery strategies in actual brain circuits. Moreover, this biological setup can become a benchmark to empirically test network theories about the spontaneous recovery in dynamical networks.

Grafted human pluripotent stem cell-derived cortical neurons integrate into adult human cortical neural circuitry.

Several neurodegenerative diseases cause loss of cortical neurons, leading to sensory, motor, and cognitive impairments. Studies in different animal models have raised the possibility that transplantation of human cortical neuronal progenitors, generated from pluripotent stem cells, might be developed into a novel therapeutic strategy for disorders affecting cerebral cortex. For example, we have shown that human long-term neuroepithelial-like stem (lt-NES) cell-derived cortical neurons, produced from induced pluripotent stem cells and transplanted into stroke-injured adult rat cortex, improve neurological deficits and establish both afferent and efferent morphological and functional connections with host cortical neurons. So far, all studies with human pluripotent stem cell-derived neurons have been carried out using xenotransplantation in animal models. Whether these neurons can integrate also into adult human brain circuitry is unknown. Here, we show that cortically fated lt-NES cells, which are able to form functional synaptic networks in cell culture, differentiate to mature, layer-specific cortical neurons when transplanted ex vivo onto organotypic cultures of adult human cortex. The grafted neurons are functional and establish both afferent and efferent synapses with adult human cortical neurons in the slices as evidenced by immuno-electron microscopy, rabies virus retrograde monosynaptic tracing, and whole-cell patch-clamp recordings. Our findings provide the first evidence that pluripotent stem cell-derived neurons can integrate into adult host neural networks also in a human-to-human grafting situation, thereby supporting their potential future clinical use to promote recovery by neuronal replacement in the patient's diseased brain.

In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology.

Recent progress in stem cell biology and epigenetic reprogramming has opened up previously unimaginable possibilities to study and develop regenerative approaches for neurological disorders. Human neurons and glial cells can be generated by differentiation of embryonic and neural stem cells and from somatic cells through reprogramming to pluripotency (followed by differentiation) as well as by direct conversion. All of these cells have the potential to be used for studying and treating neurological disorders. However, before considering using human neural cells derived from these sources for modelling or regenerative purposes, they need to be verified in terms of functionality and similarity to endogenous cells in the central nervous system (CNS).In this chapter, we describe how to assess functionality of neurons and astrocytes derived from stem cells and through direct reprogramming, using calcium imaging and electrophysiology.

Attenuation of reactive gliosis in stroke-injured mouse brain does not affect neurogenesis from grafted human iPSC-derived neural progenitors.

Induced pluripotent stem cells (iPSCs) or their progeny, derived from human somatic cells, can give rise to functional improvements after intracerebral transplantation in animal models of stroke. Previous studies have indicated that reactive gliosis, which is associated with stroke, inhibits neurogenesis from both endogenous and grafted neural stem/progenitor cells (NSPCs) of rodent origin. Here we have assessed whether reactive astrocytes affect the fate of human iPSC-derived NSPCs transplanted into stroke-injured brain. Mice with genetically attenuated reactive gliosis (deficient for GFAP and vimentin) were subjected to cortical stroke and cells were implanted adjacent to the ischemic lesion one week later. At 8 weeks after transplantation, immunohistochemical analysis showed that attenuated reactive gliosis did not affect neurogenesis or commitment towards glial lineage of the grafted NSPCs. Our findings, obtained in a human-to-mouse xenograft experiment, provide evidence that the reactive gliosis in stroke-injured brain does not affect the formation of new neurons from intracortically grafted human iPSC-derived NSPCs. However, for a potential clinical translation of these cells in stroke, it will be important to clarify whether the lack of effect of reactive gliosis on neurogenesis is observed also in a human-to-human experimental setting.

Transplantation of reprogrammed neurons for improved recovery after stroke.

Somatic cells such as fibroblasts, reprogrammed to induced pluripotent stem cells, can be used to generate neural stem/progenitor cells or neuroblasts for transplantation. In this review, we summarize recent studies demonstrating that when grafted intracerebrally in animal models of stroke, reprogrammed neurons improve function, probably by several different mechanisms, e.g., trophic actions, modulation of inflammation, promotion of angiogenesis, cellular and synaptic plasticity, and neuroprotection. In our own work, we have shown that human skin-derived reprogrammed neurons, fated to cortical progeny, integrate in stroke-injured neuronal network and form functional afferent synapses with host neurons, responding to peripheral sensory stimulation. However, whether neuronal replacement plays a role for the improvement of sensory, motor, and cognitive deficits after transplantation of reprogrammed neurons is still unclear. We conclude that further preclinical studies are needed to understand the therapeutic potential of grafted reprogrammed neurons and to define a road map for their clinical translation in stroke.