Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Human olfactory mesenchymal stromal cell transplantation ameliorates experimental autoimmune encephalomyelitis revealing an inhibitory role for IL16 on myelination.

One of the therapeutic approaches for the treatment of the autoimmune demyelinating disease, multiple sclerosis (MS) is bone marrow mesenchymal stromal cell (hBM-MSCs) transplantation. However, given their capacity to enhance myelination in vitro, we hypothesised that human olfactory mucosa-derived MSCs (hOM-MSCs) may possess additional properties suitable for CNS repair. Herein, we have examined the efficacy of hOM-MSCs versus hBM-MSCs using the experimental autoimmune encephalomyelitis (EAE) model. Both MSC types ameliorated disease, if delivered during the initial onset of symptomatic disease. Yet, only hOM-MSCs improved disease outcome if administered during established disease when animals had severe neurological deficits. Histological analysis of spinal cord lesions revealed hOM-MSC transplantation reduced blood-brain barrier disruption and inflammatory cell recruitment and enhanced axonal survival. At early time points post-hOM-MSC treatment, animals had reduced levels of circulating IL-16, which was reflected in both the ability of immune cells to secrete IL-16 and the level of IL-16 in spinal cord inflammatory lesions. Further in vitro investigation revealed an inhibitory role for IL-16 on oligodendrocyte differentiation and myelination. Moreover, the availability of bioactive IL-16 after demyelination was reduced in the presence of hOM-MSCs. Combined, our data suggests that human hOM-MSCs may have therapeutic benefit in the treatment of MS via an IL-16-mediated pathway, especially if administered during active demyelination and inflammation.

Therapeutic Potential of Niche-Specific Mesenchymal Stromal Cells for Spinal Cord Injury Repair.

The use of mesenchymal stem/stromal cells (MSCs) for transplant-mediated repair represents an important and promising therapeutic strategy after spinal cord injury (SCI). The appeal of MSCs has been fuelled by their ease of isolation, immunosuppressive properties, and low immunogenicity, alongside the large variety of available tissue sources. However, despite reported similarities in vitro, MSCs sourced from distinct tissues may not have comparable biological properties in vivo. There is accumulating evidence that stemness, plasticity, immunogenicity, and adaptability of stem cells is largely controlled by tissue niche. The extrinsic impact of cellular niche for MSC repair potential is therefore important, not least because of its impact on ex vivo expansion for therapeutic purposes. It is likely certain niche-targeted MSCs are more suited for SCI transplant-mediated repair due to their intrinsic capabilities, such as inherent neurogenic properties. In addition, the various MSC anatomical locations means that differences in harvest and culture procedures can make cross-comparison of pre-clinical data difficult. Since a clinical grade MSC product is inextricably linked with its manufacture, it is imperative that cells can be made relatively easily using appropriate materials. We discuss these issues and highlight the importance of identifying the appropriate niche-specific MSC type for SCI repair.

Multi-target approaches to CNS repair: olfactory mucosa-derived cells and heparan sulfates.

Spinal cord injury (SCI) remains one of the biggest challenges in the development of neuroregenerative therapeutics. Cell transplantation is one of numerous experimental strategies that have been identified and tested for efficacy at both preclinical and clinical levels in recent years. In this Review, we briefly discuss the state of human olfactory cell transplantation as a therapy, considering both its current clinical status and its limitations. Furthermore, we introduce a mesenchymal stromal cell derived from human olfactory tissue, which has the potential to induce multifaceted reparative effects in the environment within and surrounding the lesion. We argue that no single therapy will be sufficient to treat SCI effectively and that a combination of cell-based, rehabilitation and pharmaceutical interventions is the most promising approach to aid repair. For this reason, we also introduce a novel pharmaceutical strategy based on modifying the activity of heparan sulfate, an important regulator of a wide range of biological cell functions. The multi-target approach that is exemplified by these types of strategies will probably be necessary to optimize SCI treatment.

Spatiotemporal expansion of primary progenitor zones in the developing human cerebellum.

We present histological and molecular analyses of the developing human cerebellum from 30 days after conception to 9 months after birth. Differences in developmental patterns between humans and mice include spatiotemporal expansion of both ventricular and rhombic lip primary progenitor zones to include subventricular zones containing basal progenitors. The human rhombic lip persists longer through cerebellar development than in the mouse and undergoes morphological changes to form a progenitor pool in the posterior lobule, which is not seen in other organisms, not even in the nonhuman primate the macaque. Disruptions in human rhombic lip development are associated with posterior cerebellar vermis hypoplasia and Dandy-Walker malformation. The presence of these species-specific neural progenitor populations refines our insight into human cerebellar developmental disorders.

Spatiotemporal immune zonation of the human kidney.

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly or by cross-talk with immune cells. We used single-cell RNA sequencing to resolve the spatiotemporal immune topology of the human kidney. We reveal anatomically defined expression patterns of immune genes within the epithelial compartment, with antimicrobial peptide transcripts evident in pelvic epithelium in the mature, but not fetal, kidney. A network of tissue-resident myeloid and lymphoid immune cells was evident in both fetal and mature kidney, with postnatal acquisition of transcriptional programs that promote infection-defense capabilities. Epithelial-immune cross-talk orchestrated localization of antibacterial macrophages and neutrophils to the regions of the kidney most susceptible to infection. Overall, our study provides a global overview of how the immune landscape of the human kidney is zonated to counter the dominant immunological challenge.

Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors.

Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors.

Mapping human development at single-cell resolution.

Human development is regulated by spatiotemporally restricted molecular programmes and is pertinent to many areas of basic biology and human medicine, such as stem cell biology, reproductive medicine and childhood cancer. Mapping human development has presented significant technological, logistical and ethical challenges. The availability of established human developmental biorepositories and the advent of cutting-edge single-cell technologies provide new opportunities to study human development. Here, we present a working framework for the establishment of a human developmental cell atlas exploiting single-cell genomics and spatial analysis. We discuss how the development atlas will benefit the scientific and clinical communities to advance our understanding of basic biology, health and disease.

Human olfactory mesenchymal stromal cell transplants promote remyelination and earlier improvement in gait co-ordination after spinal cord injury.

Autologous cell transplantation is a promising strategy for repair of the injured spinal cord. Here we have studied the repair potential of mesenchymal stromal cells isolated from the human olfactory mucosa after transplantation into a rodent model of incomplete spinal cord injury. Investigation of peripheral type remyelination at the injury site using immunocytochemistry for P0, showed a more extensive distribution in transplanted compared with control animals. In addition to the typical distribution in the dorsal columns (common to all animals), in transplanted animals only, P0 immunolabelling was consistently detected in white matter lateral and ventral to the injury site. Transplanted animals also showed reduced cavitation. Several functional outcome measures including end-point electrophysiological testing of dorsal column conduction and weekly behavioural testing of BBB, weight bearing and pain, showed no difference between transplanted and control animals. However, gait analysis revealed an earlier recovery of co-ordination between forelimb and hindlimb stepping in transplanted animals. This improvement in gait may be associated with the enhanced myelination in ventral and lateral white matter, where fibre tracts important for locomotion reside. Autologous transplantation of mesenchymal stromal cells from the olfactory mucosa may therefore be therapeutically beneficial in the treatment of spinal cord injury. GLIA 2017 GLIA 2017;65:639-656.

Are nestin-positive mesenchymal stromal cells a better source of cells for CNS repair?

In recent years there has been a great deal of research within the stem cell field which has led to the definition and classification of a range of stem cells from a plethora of tissues and organs. Stem cells, by classification, are considered to be pluri- or multipotent and have both self-renewal and multi-differentiation capabilities. Presently there is a great deal of interest in stem cells isolated from both embryonic and adult tissues in the hope they hold the therapeutic key to restoring or treating damaged cells in a number of central nervous system (CNS) disorders. In this review we will discuss the role of mesenchymal stromal cells (MSCs) isolated from human olfactory mucosa, with particular emphasis on their potential role as a candidate for transplant mediated repair in the CNS. Since nestin expression defines the entire population of olfactory mucosal derived MSCs, we will compare these cells to a population of neural crest derived nestin positive population of bone marrow-MSCs.

Comparative miRNA-Based Fingerprinting Reveals Biological Differences in Human Olfactory Mucosa- and Bone-Marrow-Derived Mesenchymal Stromal Cells.

Previously we reported that nestin-positive human mesenchymal stromal cells (MSCs) derived from the olfactory mucosa (OM) enhanced CNS myelination in vitro to a greater extent than bone-marrow-derived MSCs (BM-MSCs). miRNA-based fingerprinting revealed the two MSCs were 64% homologous, with 26 miRNAs differentially expressed. We focused on miR-146a-5p and miR-140-5p due to their reported role in the regulation of chemokine production and myelination. The lower expression of miR-140-5p in OM-MSCs correlated with higher secretion of CXCL12 compared with BM-MSCs. Addition of CXCL12 and its pharmacological inhibitors to neural co-cultures supported these data. Studies on related miR-146a-5p targets demonstrated that OM-MSCs had lower levels of Toll-like receptors and secreted less pro-inflammatory cytokines, IL-6, IL-8, and CCL2. OM-MSCs polarized microglia to an anti-inflammatory phenotype, illustrating potential differences in their inflammatory response. Nestin-positive OM-MSCs could therefore offer a cell transplantation alternative for CNS repair, should these biological behaviors be translated in vivo.

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Shiga toxin associated hemolytic uremic syndrome.

Shiga toxin associated hemolytic uremic syndrome (Stx HUS), a thrombotic microangiopathy, is the most common cause of pediatric acute kidney injury but has no direct treatment. A better understanding of disease pathogenesis may help identify new therapeutic targets. For this reason, the role of complement is being actively studied while eculizumab, the C5 monoclonal antibody, is being used to treat Stx HUS but with conflicting results. A randomized controlled trial would help properly evaluate its use in Stx HUS while more research is required to fully evaluate the role complement plays in the disease pathogenesis.

Directed differentiation of umbilical cord blood stem cells into cortical GABAergic neurons.

Umbilical cord blood contains a population of non-hematopoietic multipotent stem cells that are capable of neuronal differentiation in-vitro. These cells have shown great potential as a therapeutic tool for central nervous system diseases and disorders. However whether these cells are able to produce neurons with similar developmental and functional characteristics to indigenous neurons within the brain remains poorly investigated. In this study, we used purified umbilical cord blood non-hematopoietic stem cells to produced GABAergic neurons with similar developmental and functional characteristics to cortical GABAergic neurons. We analyzed the expression of transcription factors MASH1, DLX1 and DLX2 throughout the 24 days of a sequential neuronal induction protocol and found that their expression patterns resembled those reported in the developing human cortex. The derived neurons also expressed components of GABAergic neurotransmission including GABA regulatory enzymes, GABA receptor subunits and GABA transporters. Thus we have demonstrated that umbilical cord blood stem cells are capable of producing cortical-like GABAergic neurons in vitro. This highlights the potential of umbilical cord blood stem cells as a therapeutic tool for neural injuries and disorders.

Human mesenchymal stem cells isolated from olfactory biopsies but not bone enhance CNS myelination in vitro.

Spinal cord injury (SCI) is a devastating condition with limited capacity for repair. Cell transplantation is a potential strategy to promote SCI repair with cells from the olfactory system being promising candidates. Although transplants of human olfactory mucosa (OM) are already ongoing in clinical trials, the repair potential of this tissue remains unclear. Previously, we identified mesenchymal-like stem cells that reside in the lamina propria (LP-MSCs) of rat and human OM. Little is known about these cells or their interactions with glia such as olfactory ensheathing cells (OECs), which would be co-transplanted with MSCs from the OM, or endogenous CNS glia such as oligodendrocytes. We have characterized, purified, and assessed the repair potential of human LP-MSCs by investigating their effect on glial cell biology with specific emphasis on CNS myelination in vitro. Purified LP-MSCs expressed typical bone marrow MSC (BM-MSC) markers, formed spheres, were clonogenic and differentiated into bone and fat. LP-MSC conditioned medium (CM) promoted oligodendrocyte precursor cell (OPC) and OEC proliferation and induced a highly branched morphology. LP-MSC-CM treatment caused OEC process extension. Both LP and BM-MSCs promoted OPC proliferation and differentiation, but only myelinating cultures treated with CM from LP and not BM-MSCs had a significant increase in myelination. Comparison with fibroblasts and contaminating OM fibroblast like-cells showed the promyelination effect was LP-MSC specific. Thus LP-MSCs harvested from human OM biopsies may be an important candidate for cell transplantation by contributing to the repair of SCI.

Investigating embryonic expression patterns and evolution of AHI1 and CEP290 genes, implicated in Joubert syndrome.

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man.

In vitro modelling of cortical neurogenesis by sequential induction of human umbilical cord blood stem cells.

Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.

The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis.

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.

The corticofugal neuron-associated genes ROBO1, SRGAP1, and CTIP2 exhibit an anterior to posterior gradient of expression in early fetal human neocortex development.

Developing neocortical progenitors express transcription factors in gradients that induce programs of region-specific gene expression. Our previous work identified anteriorly upregulated expression gradients of a number of corticofugal neuron-associated gene probe sets along the anterior-posterior axis of the human neocortex (8-12 postconceptional weeks [PCW]). Here, we demonstrate by real-time polymerase chain reaction, in situ hybridization and immunohistochemistry that 3 such genes, ROBO1, SRGAP1, and CTIP2 are highly expressed anteriorly between 8-12 PCW, in comparison with other genes (FEZF2, SOX5) expressed by Layer V, VI, and subplate neurons. All 3 were prominently expressed by early postmitotic neurons in the subventricular zone, intermediate zone, and cortical plate (CP) from 8 to 10 PCW. Between 12 and 15 PCW expression patterns for ER81 and SATB2 (Layer V), TBR1 (Layer V/VI) and NURR1 (Layer VI) revealed Layer V forming. By 15 PCW, ROBO1 and SRGAP1 expression was confined to Layer V, whereas CTIP2 was expressed throughout the CP anteriorly. We observed ROBO1 and SRGAP1 immunoreactivity in medullary corticospinal axons from 11 PCW onward. Thus, we propose that the coexpression of these 3 markers in the anterior neocortex may mark the early location of the human motor cortex, including its corticospinal projection neurons, allowing further study of their early differentiation.

Investigating gradients of gene expression involved in early human cortical development.

The division of the neocortex into functional areas (the cortical map) differs little between individuals, although brain lesions in development can lead to substantial re-organization of regional identity. We are studying how the cortical map is established in the human brain as a first step towards understanding this plasticity. Previous work on rodent development has identified certain transcription factors (e.g. Pax6, Emx2) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area-specific thalamocortical afferent projections. Although mechanisms may be shared, the human neocortex is composed of different and more complex local area identities. Using Affymetrix gene chips of human foetal brain tissue from 8 to 12.5 post-conceptional weeks [PCW, equivalent to Carnegie stage (CS) 23, to Foetal stage (F) 4], human material obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org), we have identified a number of genes that exhibit gradients along the anterior-posterior axis of the neocortex. Gene probe sets that were found to be upregulated posteriorally compared to anteriorally, included EMX2, COUPTFI and FGF receptor 3, and those upregulated anteriorally included cell adhesion molecules such as cadherins and protocadherins, as well as potential motor cortex markers and frontal markers (e.g. CNTNAP2, PCDH17, ROBO1, and CTIP2). Confirmation of graded expression for a subset of these genes was carried out using real-time PCR. Furthermore, we have established a dissociation cell culture model utilizing tissue dissected from anteriorally or posteriorally derived developing human neocortex that exhibits similar gradients of expression of these genes for at least 72 h in culture.