First-in-human stage III/IV melanoma​ clinical trial of immune priming agent IFx-Hu2.0.

IFx-Hu2.0 was designed to encode part of the Emm55 protein contained within a plasmid in a formulation intended for transfection into mammalian cells. IFx-Hu2.0 promotes both adaptive and innate immune responses in animal studies. Furthermore, previous studies have demonstrated safety/efficacy in equine, canine, and murine species. We present the first-in-human study of IFx-Hu2.0, administered by intralesional injection into melanoma tumors of seven patients with stage III/IV unresectable melanoma. No dose-limiting toxicities attributable to IFx-Hu2.0 were observed. Grade 1/2 injection site reactions were observed in five of seven patients. IgG and IgM responses were seen in the peripheral blood to Emm55 peptides and known melanoma antigens, suggesting that IFx-Hu2.0 acts as an individualized "in-situ vaccine." Three of four patients previously refractory to anti-PD1 experienced clinical benefit upon subsequent anti-PD1-based treatment. Therefore, this approach is feasible, and clinical/correlative outcomes warrant further investigation for treating metastatic melanoma patients as an immune priming agent.

Neurogenin 3 is regulated by neurotrophic tyrosine kinase receptor type 2 (TRKB) signaling in the adult human exocrine pancreas.

BACKGROUND: Reports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas. METHODS: Transcriptome analysis, quantitative reverse transcriptase polymerase chain reaction (RTPCR) and immunochemistry were used to identify TRKB isoform expression in primary cultures of human islet-depleted exocrine tissue and human cadaveric pancreas biopsies. The effects of pharmacological modulation of TRKB signaling on the expression of NGN3 were assessed by Student's t-test and ANOVA. RESULTS: Approximately 30 % of cultured exocrine cells and 95 % of NGN3+ cells express TRKB on their cell surface. Transcriptome-based exon splicing analyses, isoform-specific quantitative RTPCR and immunochemical staining demonstrate that TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. Pharmacological inhibition of TRKB significantly decreased the percentage of NGN3+ cells, while a TRKB agonist significantly increased this percentage. Inhibition of protein kinase B (AKT) blocked the effect of the TRKB agonist, while inhibition of tyrosine kinase had no effect. Modulation of TRKB and AKT signaling did not significantly affect the level of NGN3 mRNA. CONCLUSIONS: In the adult human exocrine pancreas, TRKB-T1 positively regulates NGN3 independent of effects on NGN3 transcription. Targeting mechanisms controlling the NGN3+ cell population size and endocrine cell fate commitment represent a potential new approach to understand pancreas pathobiology and means whereby cell populations could be expanded for therapeutic purposes.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment.

Chondroitin sulfate proteoglycans inhibit oligodendrocyte myelination through PTPσ.

CNS damage often results in demyelination of spared axons due to oligodendroglial cell death and dysfunction near the injury site. Although new oligodendroglia are generated following CNS injury and disease, the process of remyelination is typically incomplete resulting in long-term functional deficits. Chondroitin sulfate proteoglycans (CSPGs) are upregulated in CNS grey and white matter following injury and disease and are a major component of the inhibitory scar that suppresses axon regeneration. CSPG inhibition of axonal regeneration is mediated, at least in part, by the protein tyrosine phosphatase sigma (PTPσ) receptor. Recent evidence demonstrates that CSPGs inhibit OL process outgrowth, however, the means by which their effects are mediated remains unclear. Here we investigate the role of PTPσ in CSPG inhibition of OL function. We found that the CSPGs, aggrecan, neurocan and NG2 all imposed an inhibitory effect on OL process outgrowth and myelination. These inhibitory effects were reversed by degradation of CSPGs with Chondroitinase ABC prior to OL exposure. RNAi-mediated down-regulation of PTPσ reversed the inhibitory effect of CSPGs on OL process outgrowth and myelination. Likewise, CSPG inhibition of process outgrowth and myelination was significantly reduced in cultures containing PTPσ(-/-) OLs. Finally, inhibition of Rho-associated kinase (ROCK) increased OL process outgrowth and myelination during exposure to CSPGs. These results suggest that in addition to their inhibitory effects on axon regeneration, CSPGs have multiple inhibitory actions on OLs that result in incomplete remyelination following CNS injury. The identification of PTPσ as a receptor for CSPGs, and the participation of ROCK downstream of CSPG exposure, reveal potential therapeutic targets to enhance white matter repair in the damaged CNS.

Engraftment of human embryonic stem cell derived cardiomyocytes improves conduction in an arrhythmogenic in vitro model.

In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-ß treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275 ± 36 ms and conduction velocity (CV) of 10.6 ± 4.2 cm/s (n = 3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4 ± 0.9 cm/s, n = 35 vs. 30.1 ± 3.2 cm/s, n = 20 in the longitudinal direction and 4.3 ± 0.3 cm/s vs. 9.3 ± 0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6 ± 1.3 cm/s and 3.1 ± 0.5 cm/s, n = 11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.

Use of perfluorocarbon nanoparticles for non-invasive multimodal cell tracking of human pancreatic islets.

In vivo imaging of engraftment and immunorejection of transplanted islets is critical for further clinical development, with (1)H MR imaging of superparamagnetic iron oxide-labeled cells being the current premier modality. Using perfluorocarbon nanoparticles, we present here a strategy for non-invasive imaging of cells using other modalities. To this end, human cadaveric islets were labeled with rhodamine-perfluorooctylbromide (PFOB) nanoparticles, rhodamine-perfluoropolyether (PFPE) nanoparticles or Feridex as control and tested in vitro for cell viability and c-peptide secretion for 1 week. (19)F MRI, computed tomography (CT) and ultrasound (US) imaging was performed on labeled cell phantoms and on cells following transplantation beneath the kidney capsule of mice and rabbits. PFOB and PFPE-labeling did not reduce human islet viability or glucose responsiveness as compared with unlabeled cells or SPIO-labeled cells. PFOB- and PFPE-labeled islets were effectively fluorinated for visualization by (19)F MRI. PFOB-labeled islets were acoustically reflective for detection by US imaging and became sufficiently brominated to become radiopaque allowing visualization with CT. Thus, perfluorocarbon nanoparticles are multimodal cellular contrast agents that may find applications in real-time targeted delivery and imaging of transplanted human islets or other cells in a clinically applicable manner using MRI, US or CT imaging.

OCT3/4 regulates transcription of histone deacetylase 4 (Hdac4) in mouse embryonic stem cells.

OCT3/4 is a POU domain transcription factor that is critical for maintenance of pluripotency and self-renewal by embryonic stem (ES) cells and cells of the early mammalian embryo. It has been demonstrated to bind and regulate a number of genes, often in conjunction with the transcription factors SOX2 and NANOG. In an effort to further understand this regulatory network, chromatin immunoprecipitation was used to prepare a library of DNA segments specifically bound by OCT3/4 in undifferentiated mouse ES (mES) cell chromatin. One segment corresponds to a region within the first intron of the gene encoding histone deacetylase 4 (Hdac4), a Class II histone deacetylase. This region acts as a transcriptional repressor and contains at least two functional sites that are specifically bound by OCT3/4. HDAC4 is not expressed in the nuclei of OCT3/4+ mES cells and is upregulated upon differentiation. These findings demonstrate the participation of OCT3/4 in the repression of Hdac4 in ES cells.

Proliferation and pluripotency of human embryonic stem cells maintained on type I collagen.

Human embryonic stem cells (hESC) require a balance of growth factors and signaling molecules to proliferate and retain pluripotency. Conditioned medium (CM) from a human embryonic germ-cell-derived cell culture, SDEC, was observed to support the growth of hESC on type I collagen (COL I) and on Matrigel (MAT) biomatricies. After 1 month, the population doubling of hESC grown in SDEC CM on COL I was equivalent to that of hESC grown in mouse embryonic fibroblast (MEF) CM on MAT. hESC grown in SDEC CM on COL I expressed OCT4, NANOG, SSEA-4, alkaline phosphatase (AP), and TRA-1-60; retained a normal karyotype; and were capable of forming teratomas. DNA microarray analysis was used to compare the transcriptional profiles of SDEC and the less supportive WI38 and Detroit 551 human cell lines. The mRNA level of secreted frizzled-related protein (sFRP-1), a known antagonist of the WNT/ß-catenin signaling pathway, was significantly reduced in SDEC as compared with the other 2 cell lines, whereas the mRNA levels of prostaglandin-endoperoxide synthase 2 (PTGS2 or COX-2) and prostaglandin I2 synthase (PGIS), two prostaglandin biosynthesis genes, were significantly increased in SDEC. The level of sFRP-1 protein was significantly reduced, and levels of 2 prostaglandins that are downstream products of PTGS2 and PGIS, prostaglandin E2 and 6-keto-prostaglandin F(1α), were significantly elevated in SDEC CM compared with WI38, Detroit 551, and MEF CM. Further, addition of purified sFRP-1 to SDEC CM reduced the proliferation of hESC grown on COL I as well as MAT in a dose-dependent manner.

Stem cells and the liver: clinical applications in transplantation.

ESLD affects millions of Americans, and HCV is a worldwide pandemic. Unfortunately, the ability to study liver disease and novel therapeutics experimentally in the laboratory is limited by an ongoing lack of small animal models. The development of rodents with livers chimeric for human hepatocytes may improve this situation. The authors' efforts currently use an immunodeficient or exogenously immunosuppressed animal with subsequent liver injury provided by chemical or surgical means. Cell transplantation with either human hepatocytes or human stem cells results in engraftment and subsequent "humanization" of an animal liver. Study of these animal models may lead to innovative approaches to the management of ESLD in both children and adults.

Physical transfer of membrane and cytoplasmic components as a general mechanism of cell-cell communication.

Recent evidence from different research areas has revealed a novel mechanism of cell-cell communication by spontaneous intercellular transfer of cellular components (ICT). Here we studied this phenomenon by co-culturing different cells that contain distinct levels of proteins or markers for the plasma membrane or cytoplasm. We found that a variety of transmembrane proteins are transferable between multiple cell types. Membrane lipids also show a high efficiency of intercellular transfer. Size-dependent cytoplasmic transfer allows exchange of cytoplasmic macromolecules up to 40 kDa between somatic cells, and up to 2000 kDa between uncommitted human precursor cells and human umbilical vein endothelial cells. Protein transfer, lipid transfer and cytoplasmic component transfer can occur simultaneously and all require direct cell-cell contact. Analyses of the properties of ICT, together with a close examination of cell-cell interactions, suggest that the spontaneous ICT of different cellular components might have a common underlying process: transient local membrane fusions formed when neighboring cells undergo close cell-cell contact.

Embryonic germ cells are capable of adipogenic differentiation in vitro and in vivo.

There is an extensive clinical need for soft tissue filler materials, such as adipose tissue, for plastic and reconstructive surgery. Due to limitations with autologous adipose transplantation, engineered adipose tissue provides a potential alternative therapy. Embryonic germ cells form embryoid bodies and subsequent embryoid body-derived (EBD) cells have the ability to differentiate toward multiple tissue types. The objective of this study was to demonstrate that EBD cells were capable of adipogenic differentiation in vitro and in vivo using a poly(ethylene glycol)-based hydrogel scaffold. EBD cells underwent adipogenic differentiation in vitro and in vivo. Results were directly compared to adipogenic differentiation of adult bone marrow-derived mesenchymal stem cells (MSCs). Differentiated EBD cells in both monolayer and three-dimensional in vitro culture demonstrated fat granules by light microscopy, stained positive for lipids with oil red-O, and expressed adipocyte-specific genes (lipoprotein lipase [LPL], peroxisome proliferator activated receptor gamma2, and adipocyte-specific fatty acid binding protein [alphaP2]). In vivo constructs demonstrated adipogenic differentiation by alphaP2 and LPL gene expression and oil red-O staining of lipid granules. In conclusion, EBD cells are capable of differentiating toward an adipogenic lineage in vitro and in vivo. EBD cells' adipogenic differentiation is comparable to that of MSCs and demonstrate therapeutic potential for soft tissue augmentation and reconstruction.

Imprinting status of Galpha(s), NESP55, and XLalphas in cell cultures derived from human embryonic germ cells: GNAS imprinting in human embryonic germ cells.

GNAS is a complex gene that through use of alternative first exons encodes signaling proteins Galpha(s) and XLalphas plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of Galpha(s), NESP55, and XLalphas prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of Galpha(s) was biallelic (maternal allele: 52.6%+/- 2.5%; paternal allele: 47.2%+/- 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%+/- 10%; paternal allele: 18.1%+/- 10%; p= 0.002) and XLalphas was preferentially expressed from the paternal allele (maternal allele: 2.7%+/- 0.3%; paternal allele: 97.3%+/- 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5-11 weeks postfertilization, and that imprinting of XLalphas occurs very early postfertilization. By contrast, imprinting of Galpha(s) most likely occurs after 11 weeks postfertilization and after tissue differentiation.

Cells derived from human umbilical cord blood support the long-term growth of undifferentiated human embryonic stem cells.

Various types of human cells have been tested as feeder cells for the undifferentiated growth of human embryonic stem cells (hESCs) in vitro. We report here the successful culture of two hESC lines (H1 and H9) on human umbilical cord blood (UCB)-derived fibroblast-like cells. These cells permit the long-term continuous growth of undifferentiated and pluripotent hESCs. The cultured hESCs had normal karyotypes, expressed OCT-4, SSEA-4, TRA-1-60, and TRA-1-81, formed cystic embryonic body in vitro and teratomas in vivo after injected into immunodeficient mice. The wide availability of clinical-grade human UCB makes it a promising source of support cells for the growth of hESC for use in cell therapies.

Generation of humanized animal livers using embryoid body-derived stem cell transplant.

OBJECTIVE: Animal organs engineered to be chimeric for human cells could contribute significantly to the field of transplantation, including studies of human-specific diseases such as hepatitis-C, as treatment for in-born errors of metabolism, and for development of a renewable source of transplantable organs via modified xenotransplantation. We sought to use human embryoid body-derived stem cells (EBDs) to populate livers in animals for applications in transplant surgery. METHODS: SCID mice and rats underwent liver injury with carbon tetrachloride exposure or partial hepatectomy. Animals received intrasplenic injection of fluorescently labeled human stem cells. Spleen and liver were assessed at 2, 7, 15, and 30 days after transplant for the presence of EBDs and markers of human hepatocyte differentiation. RESULTS: EBDs migrate to and engraft in animal liver after splenic injection under conditions of hepatic injury. EBDs are detectable at 2 days and are in abundance at 1 week after transplant. EBDs persist in rodent liver long term (>1 month), and once engrafted differentiate into functional human hepatocytes as assessed by production of human alpha-feto-protein (AFP) and human albumin. CONCLUSIONS: We developed a novel animal model in which hepatic injury and stem cell transplantation lead to the generation of humanized animal organs. We are currently using our model to study recurrent hepatitis-C after liver transplantation, and as an alternative to whole organ transplantation for treatment of in-born errors of metabolism.

SOX17 directly activates Zfp202 transcription during in vitro endoderm differentiation.

SOX17 is a SRY-related high-mobility group (HMG) box transcription factor that is necessary for endoderm formation in multiple species. Despite its essential function during endoderm formation and differentiation, few direct targets of SOX17 are known. To identify targets of SOX17, we isolated SOX17 binding sites with a chromatin immunoprecipitation (ChIP)-cloning screen. SOX17-ChIP identified zinc finger protein 202 (Zfp202) as a direct target of SOX17 during endoderm differentiation of F9 embryonal carcinoma cells. A sequence in the first intron of Zfp202 activated transcription in differentiated F9 cells, and overexpression of Sox17 increased the transcriptional activity of this sequence. SOX17 binds to a site within this sequence in electrophoretic mobility shift assays, and mutation of this site decreases the transcriptional activation. Zfp202 is induced concomitantly with Sox17 during endoderm differentiation of F9 cells. We also show that ZFP202 represses Hnf4a, which has been reported for the human ortholog ZNF202. Identifying targets of SOX17 will help to elucidate the molecular basis of endoderm differentiation and may provide a better understanding of the role of endoderm in patterning the other germ layers.

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives.

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and beta-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

Pluripotent stem cells from germ cells.

To date, stem cells have been derived from three sources of germ cells. These include embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and multipotent germ line stem cells (GSCs). EGCs are derived from primordial germ cells that arise in the late embryonic and early fetal period of development. ECCs are derived from adult testicular tumors whereas GSCs have been derived by culturing spermatogonial stem cells from mouse neonates and adults. For each of these lines, their pluripotency has been demonstrated by their ability to differentiate into cell types derived from the three germ layers in vitro and in vivo and in chimeric animals, including germ line transmission. These germ line-derived stem cells have been generated from many species including human, mice, porcine, and chicken albeit with only slight modifications. This chapter describes general considerations regarding critical aspects of their derivation compared with their counterpart, embryonic stem cells (ESCs). Detailed protocols for EGC derivation and maintenance from human and mouse primordial germ cells (PGCs) will be presented.

Stem cell profiling by nuclear magnetic resonance spectroscopy.

The classification of embryonic and adult stem cells, including their derivatives, is still limited, and often these cells are best defined by their functional properties. Recent gene array studies have yielded contradictory results. Also, very little is known about the metabolic properties of these exciting cells. In this study, proton (1H) NMR spectroscopy was used to identify the major low-molecular-weight metabolites in murine embryonic stem cells (ESC) and their neural stem cell (NSC) derivatives. ESC are characterized by an unusually low number of NMR-detectable metabolites, high phosphocholine (PC) content, and nondetectable glycerophosphocholine (GPC). The metabolic profiles of NSC resemble glial cells and oligodendrocyte progenitors, but with considerably higher PC, GPC, and myo-inositol content. The results suggest that NMR spectroscopy in vitro can provide markers to study the effects of differentiation on cell metabolism, and potentially to assess stem cell preparations for differentiation status.

Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage.

Stem cell therapy is a hope for the treatment of some childhood neurological disorders. We examined whether human neural stem cells (hNSCs) replace lost cells in a newborn mouse model of brain damage. Excitotoxic lesions were made in neonatal mouse forebrain with the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA). QA induced apoptosis in neocortex, hippocampus, striatum, white matter, and subventricular zone. This degeneration was associated with production of cleaved caspase-3. Cells immunopositive for inducible nitric oxide synthase were present in damaged white matter and subventricular zone. Three days after injury, mice received brain parenchymal or intraventricular injections of hNSCs derived from embryonic germ (EG) cells. Human cells were prelabeled in vitro with DiD for in vivo tracking. The locations of hNSCs within the mouse brain were determined through DiD fluorescence and immunodetection of human-specific nestin and nuclear antigen 7 days after transplantation. hNSCs survived transplantation into the lesioned mouse brain, as evidenced by human cell markers and DiD fluorescence. The cells migrated away from the injection site and were found at sites of injury within the striatum, hippocampus, thalamus, and white matter tracts and at remote locations in the brain. Subsets of grafted cells expressed neuronal and glial cell markers. hNSCs restored partially the complement of striatal neurons in brain-damaged mice. We conclude that human EG cell-derived NSCs can engraft successfully into injured newborn brain, where they can survive and disseminate into the lesioned areas, differentiate into neuronal and glial cells, and replace lost neurons. (c) 2005 Wiley-Liss, Inc.