Purification and characterization of human neural stem and progenitor cells.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.

The ß-haemoglobinopathies, such as sickle cell disease and ß-thalassaemia, are caused by mutations in the ß-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure ß-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult ß-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for ß-haemoglobinopathies.

Efficient scarless genome editing in human pluripotent stem cells.

Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis.

Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.

Systemic Neutrophil Depletion Modulates the Migration and Fate of Transplanted Human Neural Stem Cells to Rescue Functional Repair.

The interaction of transplanted stem cells with local cellular and molecular cues in the host CNS microenvironment may affect the potential for repair by therapeutic cell populations. In this regard, spinal cord injury (SCI), Alzheimer's disease, and other neurological injuries and diseases all exhibit dramatic and dynamic changes to the host microenvironment over time. Previously, we reported that delayed transplantation of human CNS-derived neural stem cells (hCNS-SCns) at 9 or 30 d post-SCI (dpi) resulted in extensive donor cell migration, predominantly neuronal and oligodendrocytic donor cell differentiation, and functional locomotor improvements. Here, we report that acute transplantation of hCNS-SCns at 0 dpi resulted in localized astroglial differentiation of donor cells near the lesion epicenter and failure to produce functional improvement in an all-female immunodeficient mouse model. Critically, specific immunodepletion of neutrophils (polymorphonuclear leukocytes) blocked hCNS-SCns astroglial differentiation near the lesion epicenter and rescued the capacity of these cells to restore function. These data represent novel evidence that a host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population, and support targeting the inflammatory microenvironment in combination with cell transplantation after SCI.SIGNIFICANCE STATEMENT The interaction of transplanted cells with local cellular and molecular cues in the host microenvironment is a key variable that may shape the translation of neurotransplantation research to the clinical spinal cord injury (SCI) human population, and few studies have investigated these events. We show that the specific immunodepletion of polymorphonuclear leukocyte neutrophils using anti-Ly6G inhibits donor cell astrogliosis and rescues the capacity of a donor cell population to promote locomotor improvement after SCI. Critically, our data demonstrate novel evidence that a specific host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population.

Prospective isolation of neural stem and progenitor cells from the developing human brain.

Prospective isolation of defined cell types is critical for the functional study of stem cells, especially in primary human tissues. Here, we present a protocol for purifying 10 transcriptomically and functionally distinct neural stem and progenitor cell types from the developing human brain using fluorescence-activated cell sorting. We describe steps for tissue dissociation, staining, and cell sorting as well as downstream functional experiments for measuring clonogenicity, differentiation, and engraftment potential of purified populations. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.

A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2-Induced Glycolytic Reprogramming in Glioblastoma.

PURPOSE: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel PET tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM. EXPERIMENTAL DESIGN: [18F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/µmol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [18F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma. RESULTS: In mouse imaging studies, [18F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio of 3.6 ± 0.5. In human volunteers, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In patients with GBM, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [18F]DASA-23 was markedly elevated in GBMs compared with normal brain, and it identified a metabolic nonresponder within 1 week of treatment initiation. CONCLUSIONS: We developed and translated [18F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [18F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards.

Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.

CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells.

Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Increasing Human Neural Stem Cell Transplantation Dose Alters Oligodendroglial and Neuronal Differentiation after Spinal Cord Injury.

Multipotent human central nervous system-derived neural stem cells transplanted at doses ranging from 10,000 (low) to 500,000 (very high) cells differentiated predominantly into the oligodendroglial lineage. However, while the number of engrafted cells increased linearly in relationship to increasing dose, the proportion of oligodendrocytic cells declined. Increasing dose resulted in a plateau of engraftment, enhanced neuronal differentiation, and increased distal migration caudal to the transplantation sites. Dose had no effect on terminal sensory recovery or open-field locomotor scores. However, total human cell number and decreased oligodendroglial proportion were correlated with hindlimb girdle coupling errors. Conversely, greater oligodendroglial proportion was correlated with increased Ab step pattern, decreased swing speed, and increased paw intensity, consistent with improved recovery. These data suggest that transplant dose, and/or target niche parameters can regulate donor cell engraftment, differentiation/maturation, and lineage-specific migration profiles.

Human neural stem cell differentiation following transplantation into spinal cord injured mice: association with recovery of locomotor function.

Stem cells are under intense investigation as potential therapeutics for central nervous system (CNS) injury and disease. However, several reports have suggested that stem cells grown as neurospheres and transplanted into an injured environment preferentially differentiate into astrocytes, contributing to glial scar. Further, the relationship between functional recovery and cell transplantation has not been empirically investigated in early studies. Using severe combined immunodeficient (scid) mice to minimize xenograft rejection, we report that prospectively isolated human fetal CNS-derived stem cells grown as neurospheres (hCNS-SCns) survive, migrate and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord injured (SCI) NOD-scid mice. Only rarely do these cells differentiate into glial fibrillary acidic protein (GFAP)-positive astrocytes, with no apparent contribution to glial scar. hCNS-SCns engraftment was associated with recovery of locomotor function. After long-term engraftment and stable behavioral plateaus in recovery were achieved (4 months post-transplantation), locomotor improvements were abolished by selective ablation of human cells with diphtheria toxin (DT). These data suggest that hCNS-SCns survival is required for locomotor recovery, possibly via differentiation and integration of human cells in the mouse host or continuous supply of trophic or other support necessary for gains in host cell function.

Transplantation dose alters the dynamics of human neural stem cell engraftment, proliferation and migration after spinal cord injury.

The effect of transplantation dose on the spatiotemporal dynamics of human neural stem cell (hNSC) engraftment has not been quantitatively evaluated in the central nervous system. We investigated changes over time in engraftment/survival, proliferation, and migration of multipotent human central nervous system-derived neural stem cells (hCNS-SCns) transplanted at doses ranging from 10,000 to 500,000 cells in spinal cord injured immunodeficient mice. Transplant dose was inversely correlated with measures of donor cell proliferation at 2 weeks post-transplant (WPT) and dose-normalized engraftment at 16 WPT. Critically, mice receiving the highest cell dose exhibited an engraftment plateau, in which the total number of engrafted human cells never exceeded the initial dose. These data suggest that donor cell expansion was inversely regulated by target niche parameters and/or transplantation density. Investigation of the response of donor cells to the host microenvironment should be a key variable in defining target cell dose in pre-clinical models of CNS disease and injury.

An fMRI study of tool-use gestures: body part as object and pantomime.

A body part as object (BPO) gesture is one of the error patterns in apraxia. In the BPO gesture, people represent objects by their hands. To clarify the neuronal background of the BPO gesture, we compared the brain activation during the BPO gesture with that during ordinary pantomime in normal subjects using functional magnetic resonance imaging. Both the BPO gesture and the pantomime induced activation in the left parietal areas (Brodmann's area (BA) 7, 40), irrespective of the hand used. These areas might be activated by a common process of tool-related gestures. The BPO gesture also activated the right supramarginal gyrus (BA 40). This activation might reflect the characteristic process of BPO, the correlation of hands with tools by their forms and movements.

Method for purification of human hematopoietic stem cells by flow cytometry.

Human hematopoietic stem cells (HSCs) and progenitors can be isolated by enriching for a rare cell population with a combination of monoclonal antibodies (MAbs). Such an isolation scheme involves multi-step procedures including ficoll-density fractionation and presort enrichment followed by cell sorting. Over the past decade, various cell-surface and metabolic markers have been identified and used to isolate human HSCs and progenitors as summarized in Table 1. Among them, CD34 has become the most critical cell-surface marker for positively selecting a rare cell population (1,2). Within the CD34(+) cell population, the differential expression of Thy-1, CD38, and AC133 have been used to fractionate HSCs and progenitors. In order to subfractionate CD34(+) cells by these markers, the cells can be further purified by flow cytometry. HSCs can be further enriched into a Thy-1(+) (3-7), CD38(-lo) (8-10), Thy-1+ CD38(-lo) (11), or AC133+ (12,13) fraction of CD34(+) cells. Table 1 Commercially Available Cell-Surface and Metabolic Markers for Isolation of Human HSC and Progenitor Cells Marker Expression/Remark Fluorochrome conjugate recommended Reference Positive marker CD34 Positive FITC, PE, APC, BIO 1,2,33 Thy-1 Positive PE, BIO 3,4 AC133 Positive PE 12,34 Negative/low marker CD38 Negative /low FITC, PE, APC 8,9 HLA-DR( a ) Negative to low FITC, PE 35,36 Mature lineage marker, Lin- CD2 T-cell lineage FITC, PE, BIO 3 CD3 T-cell lineage FITC, PE, APC, BIO 3 CD19 B-cell lineage FITC, PE, APC, BIO 3 CD16 NK-cell lineage FITC, PE, APC, BIO 3 CD14 Myeloid lineage FITC, PE 3 CD15 Myeloid lineage FITC, PE 3 Glycophorin A Erythroid lineage FITC, PE 3 2nd Step reagent Avidin/Streptavidin For BIO MAb FITC, PE, APC, TXRD, PharRed, Cy-chrome( d ) Metabolic marker( b ) Rhodamine 123( c ) Low Mitochondria-binding dye 37,38 Hoechst 33342( c ) Low DNA-binding dye 39,40 Pyronin Y Low RNA-binding dye 39,40 Propidium iodide Negative to low Dead-cell exclusion Abbreviations: FBM, fetal bone marrow; MPB, mobilized peripheral blood; ABM, adult bone marrow; HSC, hematopoietic stem cells; FITC, fluorescein; PE, phycoerythrin; APC, allophycocyanin; TXRD, Texas red; BIO, biotinylated. ( a ) FBM, MPB HSCs express HLA-DR (41,42). ( b ) To isolate quiescent HSC. ( c ) Substrates for p-glycoprotein, encoded by MDR-1. HSC possess high levels of p-glycoprotein efflux activity. ( d ) Recommended for single laser flow cytometry only, lineage marker positive and PI positive cells can be excluded simultaneously.

[The development of children's thinking strategies: the retrieval of alternatives based on the categorization with conditional reasoning tasks].

This study examined when children are able to solve conditional reasoning problems correctly using the thinking strategy that spontaneously retrieves alternatives based on the inclusive/hierarchical relations of categories and how the strategy changes with aging. In the experiment, a total of 210 3-, 4-, 5-, 8-, 10-, 12-years-old, and adults were given the conditional reasoning problems and were also asked to justify their judgments. The result showed that 5-year-old children could solve the problems as well as the adults and they used the strategy similar to the adults, but the strategy did not necessarily continue to be used from children to adults.

Injury to the spinal cord niche alters the engraftment dynamics of human neural stem cells.

The microenvironment is a critical mediator of stem cell survival, proliferation, migration, and differentiation. The majority of preclinical studies involving transplantation of neural stem cells (NSCs) into the CNS have focused on injured or degenerating microenvironments, leaving a dearth of information as to how NSCs differentially respond to intact versus damaged CNS. Furthermore, single, terminal histological endpoints predominate, providing limited insight into the spatiotemporal dynamics of NSC engraftment and migration. We investigated the early and long-term engraftment dynamics of human CNS stem cells propagated as neurospheres (hCNS-SCns) following transplantation into uninjured versus subacutely injured spinal cords of immunodeficient NOD-scid mice. We stereologically quantified engraftment, survival, proliferation, migration, and differentiation at 1, 7, 14, 28, and 98 days posttransplantation, and identified injury-dependent alterations. Notably, the injured microenvironment decreased hCNS-SCns survival, delayed and altered the location of proliferation, influenced both total and fate-specific migration, and promoted oligodendrocyte maturation.

Clinical translation of human neural stem cells.

Human neural stem cell transplants have potential as therapeutic candidates to treat a vast number of disorders of the central nervous system (CNS). StemCells, Inc. has purified human neural stem cells and developed culture conditions for expansion and banking that preserve their unique biological properties. The biological activity of these human central nervous system stem cells (HuCNS-SC®) has been analyzed extensively in vitro and in vivo. When formulated for transplantation, the expanded and cryopreserved banked cells maintain their stem cell phenotype, self-renew and generate mature oligodendrocytes, neurons and astrocytes, cells normally found in the CNS. In this overview, the rationale and supporting data for pursuing neuroprotective strategies and clinical translation in the three components of the CNS (brain, spinal cord and eye) are described. A phase I trial for a rare myelin disorder and phase I/II trial for spinal cord injury are providing intriguing data relevant to the biological properties of neural stem cells, and the early clinical outcomes compel further development.

Safety of human neural stem cell transplantation in chronic spinal cord injury.

The spinal cord injury (SCI) microenvironment undergoes dynamic changes over time, which could potentially affect survival or differentiation of cells in early versus delayed transplantation study designs. Accordingly, assessment of safety parameters, including cell survival, migration, fate, sensory fiber sprouting, and behavioral measures of pain sensitivity in animals receiving transplants during the chronic postinjury period is required for establishing a potential therapeutic window. The goal of the study was assessment of safety parameters for delayed transplantation of human central nervous system-derived neural stem cells (hCNS-SCns) by comparing hCNS-SCns transplantation in the subacute period, 9 days postinjury (DPI), versus the chronic period, 60 DPI, in contusion-injured athymic nude rats. Although the number of surviving human cells after chronic transplantation was lower, no changes in cell migration were detected between the 9 and 60 DPI cohorts; however, the data suggest chronic transplantation may have enhanced the generation of mature oligodendrocytes. The timing of transplantation did not induce changes in allodynia or hyperalgesia measures. Together, these data support the safety of hCNS-SCns transplantation in the chronic period post-SCI.

Safety of epicenter versus intact parenchyma as a transplantation site for human neural stem cells for spinal cord injury therapy.

Neural stem cell transplantation may have the potential to yield repair and recovery of function in central nervous system injury and disease, including spinal cord injury (SCI). Multiple pathological processes are initiated at the epicenter of a traumatic spinal cord injury; these are generally thought to make the epicenter a particularly hostile microenvironment. Conversely, the injury epicenter is an appealing potential site of therapeutic human central nervous system-derived neural stem cell (hCNS-SCns) transplantation because of both its surgical accessibility and the avoidance of spared spinal cord tissue. In this study, we compared hCNS-SCns transplantation into the SCI epicenter (EPI) versus intact rostral/caudal (R/C) parenchyma in contusion-injured athymic nude rats, and assessed the cell survival, differentiation, and migration. Regardless of transplantation site, hCNS-SCns survived and proliferated; however, the total number of hCNS-SCns quantified in the R/C transplant animals was twice that in the EPI animals, demonstrating increased overall engraftment. Migration and fate profile were unaffected by transplantation site. However, although transplantation site did not alter the proportion of human astrocytes, EPI transplantation shifted the localization of these cells and exhibited a correlation with calcitonin gene-related peptide fiber sprouting. Critically, no changes in mechanical allodynia or thermal hyperalgesia were observed. Taken together, these data suggest that the intact parenchyma may be a more favorable transplantation site than the injury epicenter in the subacute period post-SCI.