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Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation.

BackgroundTo evaluate whether subretinal or intravitreal injection of human CD34+ bone marrow-derived stem cells (BMSC) can have protective effects on retinal degeneration that may be enhanced by coadministration of exosomes harvested from human bone marrow mesenchymal stem cells (MSCs).MethodsHuman CD34+ cells were harvested from the mononuclear cell fraction of bone marrow using magnetic beads and labeled with EGFP. Exosomes were harvested from cultured human MSCs under hypoxic conditions. Royal College of Surgeons (RCS) 3-weeks-old rats, immunosuppressed with cyclosporine A, received subretinal or intravitreal injection of CD34+ cells (50,000 cells), CD34+ cells with exosomes (50,000 cells+10 µg), exosomes alone (10 µg), or PBS. Retinal function was examined using electroretinography (ERG), and the eyes were harvested for histologic and immunohistochemical analysis.ResultsThe b-wave amplitude of ERG at 2 weeks after injection was significantly higher in eyes with subretinal or intravitreal CD34+ BMSC alone or in combination with exosomes when compared to PBS injected eyes or untreated contralateral eyes. At 4 weeks after injection, the ERG signal decreased in all groups but eyes with subretinal CD34+ BMSCs alone or combined with exosomes showed partially preserved ERG signal and preservation of the outer nuclear layer of the retina near the injection site on histology when compared to eyes with PBS injection. Immunohistochemical analysis identified the human cells in the outer retina. Subretinal or intravitreal exosome injection had no effect on retinal degeneration when administered alone or in combination with CD34+ cells.ConclusionsBoth subretinal and intravitreal injection of human CD34+ BMSCs can provide functional rescue of degenerating retina, although the effects were attenuated over time in this rat model. Regional preservation of the outer retina can occur near the subretinal injection site of CD34+ cells. These results suggest that CD34+ cells may have therapeutic potential in retinal degeneration.

BackgroundDiabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34+ bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34+ BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy.MethodsStreptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34+ BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers.ResultsThree distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34+ BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values

Huntington's disease (HD) is a fatal autosomal-dominant neurodegenerative disease caused by a trinucleotide CAG repeat expansion of the huntingtin gene (HTT) that affects 1 in every 10 000 individuals in the United States. Our lab developed a novel immune deficient HD mouse strain, the YACNSG, from a commonly used line, the YAC128 mouse, to enable transplantation studies using engineered human cells in addition to studying the impact of the immune system on disease progression. The primary goal of this project was to characterize this novel immune deQficient HD mouse model, using behavioral assays and histology to compare this new model to the immune competent YAC128 and immune deficient mice that had engraftment of a human immune system. Flow cytometry was used to confirm that the YACNSG strain lacked immune cells, and in vivo imaging was used to assess human mesenchymal stem/stromal cell (MSC) retention compared with a commonly used immune deficient line, the NSG mouse. We found that YACNSG were able to retain human MSCs longer than the immune competent YAC128 mice. We performed behavioral assessments starting at 4 months of age and continued testing monthly until 12 months on the accelerod and in the open field. At 12 months, brains were isolated and evaluated using immunohistochemistry for striatal volume. Results from these studies suggest that the novel immune deficient YACNSG strain of mice could provide a good model for human stem-cell based therapies and that the immune system appears to play an important role in the pathology of HD.

Protein Phosphatase 2 Regulatory Subunit B' Delta (PPP2R5D)-related intellectual disability (ID) and neurodevelopmental delay results from germline de novo mutations in the PPP2R5D gene. This gene encodes the protein PPP2R5D (also known as the B56 delta subunit), which is an isoform of the subunit family B56 of the enzyme serine/threonine-protein phosphatase 2A (PP2A). Clinical signs include intellectual disability (ID); autism spectrum disorder (ASD); epilepsy; speech problems; behavioral challenges; and ophthalmologic, skeletal, endocrine, cardiac, and genital malformations. The association of defective PP2A activity in the brain with a wide range of severity of ID, along with its role in ASD, Alzheimer's disease, and Parkinson's-like symptoms, have recently generated the impetus for further research into mutations within this gene. PP2A, together with protein phosphatase 1 (PP1), accounts for more than 90% of all phospho-serine/threonine dephosphorylations in different tissues. The specificity for a wide variety of substrates is determined through nearly 100 different PP2A holoenzymes that are formed by at least 23 types of regulatory B subunits, and two isoforms each of the catalytic subunit C and the structural subunit A. In the mammalian brain, PP2A-mediated protein dephosphorylation plays an important role in learning and memory. The PPP2R5D subunit is highly expressed in the brain and the PPP2A-PPP2R5D holoenzyme plays an important role in maintaining neurons and regulating neuronal signaling. From 2015 to 2017, 25 individuals with PPP2R5D-related developmental disorder were diagnosed. Since then, Whole-Exome Sequencing (WES) has helped to identify more unrelated individuals clinically diagnosed with a neurodevelopmental disorder with pathological variants of PPP2R5D. In this review, we discuss the current understanding of the clinical and genetic aspects of the disorder in the context of the known functions of the PP2A-PPP2R5D holoenzyme in the brain, as well as the pathogenic mutations in PPP2R5D that lead to deficient PP2A-PPP2R5D dephosphorylation and their implications during development and in the etiology of autism, Parkinson's disease, Alzheimer's disease, and so forth. In the future, tools such as transgenic animals carrying pathogenic PPP2R5D mutations, and patient-derived induced pluripotent stem cell lines need to be developed in order to fully understand the effects of these mutations on different neural cell types.

Diabetic retinopathy remains the leading cause of blindness among working-age adults in  the United States. Vision loss occurs because diabetes damages the retinal vessels. Currentl, retinal laser treatment and anti-VEGF injection therapy are availalbe to reduce vision loss from bleeding and retinal swelling associted with diabetic retinopathy. However, no treatment reverses the retinal vascular damage associated with diabetic retinopathy.

Human CD34 + stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34 + stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34 + BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34 + cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34 + BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34 + BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34 + BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34 + BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34 + BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34 + BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously, iPSCs from the same patient were differentiated into ECs. On day 34, the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54, and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible.

PurposeIntravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model.MethodsC3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina.ResultsIn vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis.ConclusionsIntravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration.

Huntington's disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, and some forms of Parkinson's disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats. Although pathogenesis has been attributed to this polyglutamine expansion, the underlying mechanisms through which the huntingtin protein functions have yet to be elucidated. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. For allele-specific targeting, transcription activator-like effectors (TALE) were designed to target single-nucleotide polymorphisms (SNP) in the mutant allele and packaged into a vector backbone containing KRAB to promote transcriptional repression of the disease-associated allele. Additional TALEs were packaged into a vector backbone containing heterodimeric FokI and were designed to be used as nucleases (TALEN) to cause a CAG-collapse in the mutant allele. Human HD fibroblasts were treated with each TALE-SNP or TALEN. Allele-expression was measured using a SNP-genotyping assay and mutant protein aggregation was quantified with Western blots for anti-ubiquitin. The TALE-SNP and TALEN significantly reduced mutant allele expression (p

BackgroundThe dura mater can be easily biopsied during most cranial neurosurgical operations. We describe a protocol that allows for robust generation of induced pluripotent stem cells (iPSCs) and neural progenitors from acutely harvested dura mater.ObjectiveTo generate iPSCs and neural progenitor cells from dura mater obtained during ventriculoperitoneal shunt surgery.MethodsDura was obtained during ventriculoperitoneal shunt surgery for normal pressure hydrocephalus from a 60-year-old patient with severe cognitive impairment. Fibroblasts were isolated from the dural matrix and transduced with nonintegrating Sendai virus for iPSC induction. A subset of successfully generated iPSC clones underwent immunocytochemical analysis, teratoma assay, karyotyping, and targeted neural differentiation.ResultsEleven iPSC clones were obtained from the transduction of an estimated 600,000 dural fibroblasts after 3 passages. Three clones underwent immunocytochemical analysis and were shown to express the transcription factors OCT-4, SOX2, and the embryonic cell markers SSEA-4, TRA-1-60, and Nanog. Two clones were tested for pluripotency and formed teratomas at the injection site in immunodeficient mice. Three clones underwent chromosomal analysis and were found to have a normal metaphase spread and karyotype. One clone underwent targeted neural differentiation and formed neural rosettes as well as TuJ1/SOX1-positive neural progenitor cells.ConclusionsIPSCs and neural progenitor cells can be efficiently derived from the dura of patients who need to undergo cranial neurosurgical operations. IPSCs were obtained with a nonintegrating virus and exhibited a normal karyotype, making them candidates for future autotransplantation after targeted differentiation to treat functional deficits.

There is much interest in the use of mesenchymal stem cells/marrow stromal cells (MSC) to treat neurodegenerative disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease. MSC present a promising tool for cell therapy and are currently being tested in FDA-approved phase I-III clinical trials for many disorders. In preclinical studies of neurodegenerative disorders, MSC have demonstrated efficacy, when used as delivery vehicles for neural growth factors. A number of investigators have examined the potential benefits of innate MSC-secreted trophic support and augmented growth factors to support injured neurons. These include overexpression of brain-derived neurotrophic factor and glial-derived neurotrophic factor, using genetically engineered MSC as a vehicle to deliver the cytokines directly into the microenvironment. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals, and regulate inflammation. These abilities are primarily modulated through paracrine actions. Clinical trials for MSC injection into the central nervous system to treat amyotrophic lateral sclerosis, traumatic brain injury, and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of Huntington's disease is discussed.

Background: Diabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34 + bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34 + BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy., Methods: Streptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34 + BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers., Results: Three distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34 + BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values <0.05 using ANOVA followed by post-hoc tests). For the intermediate and deep plexus layers, the difference was not statistically significant., Conclusions: The protective effect of intravitreal injection of the human CD34 + BMSCs on the superficial retinal capillary plexus layers is demonstrated using confocal microscopy in this murine model of diabetic retinopathy., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/atm-20-3930). The series “Novel Tools and Therapies for Ocular Regeneration” was commissioned by the editorial office without any funding or sponsorship. Susanna S. Park served as the unpaid Guest Editor of the series and serves as an unpaid editorial board member of Annals of Translational Medicine from Oct 2019 to Sep 2021, and reports grants from Allergan, grants from Roche/Novartis, outside the submitted work. The authors have no other conflicts of interest to declare., (2021 Annals of Translational Medicine. All rights reserved.)