Human monocyte subtype expression of neuroinflammation and regeneration-related genes is linked to age and sex.

Stroke is a leading cause of disability and the third cause of death. The immune system plays an essential role in post-stroke recovery. After an ischemic stroke, monocytes infiltrate the injured brain tissue and can exacerbate or mitigate the damage. Ischemic stroke is more prevalent in the aged population, and the aging brain exhibits an altered immune response. There are also sex disparities in ischemic stroke incidence, outcomes, and recovery, and these differences may be hormone-driven and determined by genetic and epigenetic factors. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of neuronal inflammation- and regeneration-related genes depends on age and sex. A FACS analysis of blood samples from 44 volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of natural killer cells increased in females. The proportion of B-cells decreased in both sexes with age, and subtypes of monocytes were not linked to age or sex. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly, ANXA1 and CD36 showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1ß, S100A8, TNFα, CD64, CD33, TGFß1, TLR8, CD91) were differentially changed in monocyte subtypes with increased aging. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes.

LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification.

The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

Oligodendrocytes in human induced pluripotent stem cell-derived cortical grafts remyelinate adult rat and human cortical neurons.

Neuronal loss and axonal demyelination underlie long-term functional impairments in patients affected by brain disorders such as ischemic stroke. Stem cell-based approaches reconstructing and remyelinating brain neural circuitry, leading to recovery, are highly warranted. Here, we demonstrate the in vitro and in vivo production of myelinating oligodendrocytes from a human induced pluripotent stem cell (iPSC)-derived long-term neuroepithelial stem (lt-NES) cell line, which also gives rise to neurons with the capacity to integrate into stroke-injured, adult rat cortical networks. Most importantly, the generated oligodendrocytes survive and form myelin-ensheathing human axons in the host tissue after grafting onto adult human cortical organotypic cultures. This lt-NES cell line is the first human stem cell source that, after intracerebral delivery, can repair both injured neural circuitries and demyelinated axons. Our findings provide supportive evidence for the potential future use of human iPSC-derived cell lines to promote effective clinical recovery following brain injuries.

MafB-dependent neurotransmitter signaling promotes ß cell migration in the developing pancreas.

Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs ß cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse ß cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced ß cell migration towards autonomic nerves and impaired ß cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.

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.

Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.

BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development. RESULTS: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest. CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.

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.

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.

Loss of Cxcr5 alters neuroblast proliferation and migration in the aged brain.

Neurogenesis, the production of new neurons from neural stem cells, dramatically decreases during aging concomitantly with increased inflammation both systemically and in the brain. However, the precise role of inflammation and whether local or systemic factors drive the neurogenic decline during aging is poorly understood. Here, we identify CXCR5/5/CXCL13 signaling as a novel regulator of neurogenesis in the aged brain. The chemokine Cxcl13 was found to be upregulated in the brain during aging. Loss of its receptor, Cxcr5, led to increased proliferation and decreased numbers of neuroblasts in the aged subventricular zone (SVZ), together with accumulation of neuroblasts in the rostral migratory stream and olfactory bulb (OB), without increasing the amount of new mature neurons in the OB. The effect on proliferation and migration was specific to neuroblasts and likely mediated through increased levels of systemic IL-6 and local Cxcl12 expression in the SVZ. Our study raises the possibility of a new mechanism by which interplay between systemic and local alterations in inflammation regulates neurogenesis during aging.

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.

Increased FUS levels in astrocytes leads to astrocyte and microglia activation and neuronal death.

Mutations of Fused in sarcoma (FUS), a ribonucleoprotein involved in RNA metabolism, have been found associated with both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Notably, besides mutations in the coding sequence, also mutations into the 3' untranslated region, leading to increased levels of the wild-type protein, have been associated with neuronal death and ALS pathology, in ALS models and patients. The mechanistic link between altered FUS levels and ALS-related neurodegeneration is far to be elucidated, as well as the consequences of elevated FUS levels in the modulation of the inflammatory response sustained by glial cells, a well-recognized player in ALS progression. Here, we studied the effect of wild-type FUS overexpression on the responsiveness of mouse and human neural progenitor-derived astrocytes to a pro-inflammatory stimulus (IL1ß) used to mimic an inflammatory environment. We found that astrocytes with increased FUS levels were more sensitive to IL1ß, as shown by their enhanced expression of inflammatory genes, compared with control astrocytes. Moreover, astrocytes overexpressing FUS promoted neuronal cell death and pro-inflammatory microglia activation. We conclude that overexpression of wild-type FUS intrinsically affects astrocyte reactivity and drives their properties toward pro-inflammatory and neurotoxic functions, suggesting that a non-cell autonomous mechanism can support neurodegeneration in FUS-mutated animals and patients.

Author Correction: Rapid and efficient induction of functional astrocytes from human pluripotent stem cells.

In the version of Supplementary Fig. 1 originally published with this paper, some images in panel e were accidental duplicates of images in panel b. This error has been corrected in the online integrated supplementary information and in the Supplementary Information PDF.

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

Rapid and efficient induction of functional astrocytes from human pluripotent stem cells.

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.

Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks.

BACKGROUND: Human fibroblasts can be directly converted to several subtypes of neurons, but cortical projection neurons have not been generated. METHODS: Here we screened for transcription factor combinations that could potentially convert human fibroblasts to functional excitatory cortical neurons. The induced cortical (iCtx) cells were analyzed for cortical neuronal identity using immunocytochemistry, single-cell quantitative polymerase chain reaction (qPCR), electrophysiology, and their ability to integrate into human neural networks in vitro and ex vivo using electrophysiology and rabies virus tracing. RESULTS: We show that a combination of three transcription factors, BRN2, MYT1L, and FEZF2, have the ability to directly convert human fibroblasts to functional excitatory cortical neurons. The conversion efficiency was increased to about 16% by treatment with small molecules and microRNAs. The iCtx cells exhibited electrophysiological properties of functional neurons, had pyramidal-like cell morphology, and expressed key cortical projection neuronal markers. Single-cell analysis of iCtx cells revealed a complex gene expression profile, a subpopulation of them displaying a molecular signature closely resembling that of human fetal primary cortical neurons. The iCtx cells received synaptic inputs from co-cultured human fetal primary cortical neurons, contained spines, and expressed the postsynaptic excitatory scaffold protein PSD95. When transplanted ex vivo to organotypic cultures of adult human cerebral cortex, the iCtx cells exhibited morphological and electrophysiological properties of mature neurons, integrated structurally into the cortical tissue, and received synaptic inputs from adult human neurons. CONCLUSIONS: Our findings indicate that functional excitatory cortical neurons, generated here for the first time by direct conversion of human somatic cells, have the capacity for synaptic integration into adult human cortex.

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.

Monocyte depletion early after stroke promotes neurogenesis from endogenous neural stem cells in adult brain.

Ischemic stroke, caused by middle cerebral artery occlusion, leads to long-lasting formation of new striatal neurons from neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of adult rodents. Concomitantly with this neurogenic response, SVZ exhibits activation of resident microglia and infiltrating monocytes. Here we show that depletion of circulating monocytes, using the anti-CCR2 antibody MC-21 during the first week after stroke, enhances striatal neurogenesis at one week post-insult, most likely by increasing short-term survival of the newly formed neuroblasts in the SVZ and adjacent striatum. Blocking monocyte recruitment did not alter the volume of the ischemic lesion but gave rise to reduced astrocyte activation in SVZ and adjacent striatum, which could contribute to the improved neuroblast survival. A similar decrease of astrocyte activation was found in and around human induced pluripotent stem cell (iPSC)-derived NSPCs transplanted into striatum at one week after stroke in monocyte-depleted mice. However, there was no effect on neurogenesis in the graft as determined 8weeks after implantation. Our findings demonstrate, for the first time, that a specific cellular component of the early inflammatory reaction in SVZ and adjacent striatum following stroke, i.e., infiltrating monocytes, compromises the short-term neurogenic response neurogenesis from endogenous NSPCs.

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.

Inflammation without neuronal death triggers striatal neurogenesis comparable to stroke.

Ischemic stroke triggers neurogenesis from neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) and migration of newly formed neuroblasts toward the damaged striatum where they differentiate to mature neurons. Whether it is the injury per se or the associated inflammation that gives rise to this endogenous neurogenic response is unknown. Here we showed that inflammation without corresponding neuronal loss caused by intrastriatal lipopolysaccharide (LPS) injection leads to striatal neurogenesis in rats comparable to that after a 30 min middle cerebral artery occlusion, as characterized by striatal DCX+ neuroblast recruitment and mature NeuN+/BrdU+ neuron formation. Using global gene expression analysis, changes in several factors that could potentially regulate striatal neurogenesis were identified in microglia sorted from SVZ and striatum of LPS-injected and stroke-subjected rats. Among the upregulated factors, one chemokine, CXCL13, was found to promote neuroblast migration from neonatal mouse SVZ explants in vitro. However, neuroblast migration to the striatum was not affected in constitutive CXCL13 receptor CXCR5(-/-) mice subjected to stroke. Infarct volume and pro-inflammatory M1 microglia/macrophage density were increased in CXCR5(-/-) mice, suggesting that microglia-derived CXCL13, acting through CXCR5, might be involved in neuroprotection following stroke. Our findings raise the possibility that the inflammation accompanying an ischemic insult is the major inducer of striatal neurogenesis after stroke.

Human induced pluripotent stem cells improve recovery in stroke-injured aged rats.

PURPOSE: Induced pluripotent stem cells (iPSCs) improve behavior and form neurons after implantation into the stroke-injured adult rodent brain. How the aged brain responds to grafted iPSCs is unknown. We determined survival and differentiation of grafted human fibroblast-derived iPSCs and their ability to improve recovery in aged rats after stroke. METHODS: Twenty-four months old rats were subjected to 30 min distal middle cerebral artery occlusion causing neocortical damage. After 48 h, animals were transplanted intracortically with human iPSC-derived long-term neuroepithelial-like stem (hiPSC-lt-NES) cells. Controls were subjected to stroke and were vehicle-injected. RESULTS: Cell-grafted animals performed better than vehicle-injected recipients in cylinder test at 4 and 7 weeks. At 8 weeks, cell proliferation was low (0.7 %) and number of hiPSC-lt-NES cells corresponded to 49.2% of that of implanted cells. Transplanted cells expressed markers of neuroblasts and mature and GABAergic neurons. Cell-grafted rats exhibited less activated microglia/macrophages in injured cortex and neuronal loss was mitigated. CONCLUSIONS: Our study provides the first evidence that grafted human iPSCs survive, differentiate to neurons and ameliorate functional deficits in stroke-injured aged brain.