Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells.

We have previously shown that human parthenogenetic stem cells (hpSC) can be chemically directed to differentiate into a homogeneous population of multipotent neural stem cells (hpNSC) that are scalable, cryopreservable, express all the appropriate neural markers, and can be further differentiated into functional dopaminergic neurons. Differentiation of hpSC into hpNSC provides a platform to study the molecular basis of human neural differentiation, to develop cell culture models of neural disease, and to provide neural stem cells for the treatment of neurodegenerative diseases. Additionally, the hpNSC that are generated could serve as a platform for drug discovery and the determination of pharmaceutical-induced neural toxicity. Here, we describe in detail the stepwise protocol that was developed in our laboratory that facilitates the highly efficient and reproducible differentiation of hpSC into hpNSC.

Human parthenogenetic neural stem cell grafts promote multiple regenerative processes in a traumatic brain injury model.

International Stem Cell Corporation human parthenogenetic neural stem cells (ISC-hpNSC) have potential therapeutic value for patients suffering from traumatic brain injury (TBI). Here, we demonstrate the behavioral and histological effects of transplanting ISC-hpNSC intracerebrally in an animal model of TBI. Methods: Sprague-Dawley rats underwent a moderate controlled cortical impact TBI surgery. Transplantation occurred at 72 h post-TBI with functional readouts of behavioral and histological deficits conducted during the subsequent 3-month period after TBI. We characterized locomotor, neurological, and cognitive performance at baseline (before TBI), then on days 0, 1, 7, 14, 30, 60, and 90 (locomotor and neurological), and on days 28-30, 58-60, and 88-90 (cognitive) after TBI. Following completion of behavioral testing at 3 months post-TBI, animals were euthanized by transcardial perfusion and brains harvested to histologically characterize the extent of brain damage. Neuronal survival was revealed by Nissl staining, and stem cell engraftment and host tissue repair mechanisms such as the anti-inflammatory response in peri-TBI lesion areas were examined by immunohistochemical analyses. Results: We observed that TBI groups given high and moderate doses of ISC-hpNSC had an improved swing bias on an elevated body swing test for motor function, increased scores on forelimb akinesia and paw grasp neurological tests, and committed significantly fewer errors on a radial arm water maze test for cognition. Furthermore, histological analyses indicated that high and moderate doses of stem cells increased the expression of phenotypic markers related to the neural lineage and myelination and decreased reactive gliosis and inflammation in the brain, increased neuronal survival in the peri-impact area of the cortex, and decreased inflammation in the spleen at 90 days post-TBI. Conclusion: These results provide evidence that high and moderate doses of ISC-hpNSC ameliorate TBI-associated histological alterations and motor, neurological, and cognitive deficits.

Novel Approach to Stem Cell Therapy in Parkinson's Disease.

In this commentary we discuss International Stem Cell Corporation's (ISCO's) approach to developing a pluripotent stem cell based treatment for Parkinson's disease (PD). In 2016, ISCO received approval to conduct the world's first clinical study of a pluripotent stem cell based therapy for PD. The Australian regulatory agency Therapeutic Goods Administration (TGA) and the Melbourne Health's Human Research Ethics Committee (HREC) independently reviewed ISCO's extensive preclinical data and granted approval for the evaluation of a novel human parthenogenetic derived neural stem cell (NSC) line, ISC-hpNSC, in a PD phase 1 clinical trial ( ClinicalTrials.gov NCT02452723). This is a single-center, open label, dose escalating 12-month study with a 5-year follow-up evaluating a number of objective and patient-reported safety and efficacy measures. A total of 6 years of safety and efficacy data will be collected from each patient. Twelve participants are recruited in this study with four participants per single dose cohort of 30, 50, and 70 million ISC-hpNSC. The grafts are placed bilaterally in the caudate nucleus, putamen, and substantia nigra by magnetic resonance imaging-guided stereotactic surgery. Participants are 30-70 years old with idiopathic PD ≤13 years duration and unified PD rating scale motor score (Part III) in the "OFF" state ≤49. This trial is fully funded by ISCO with no economic involvement from the patients. It is worth noting that ISCO underwent an exhaustive review process and successfully answered the very comprehensive, detailed, and specific questions posed by the TGA and HREC. The regulatory/ethic review process is based on applying scientific and clinical expertise to decision-making, to ensure that the benefits to consumers outweigh any risks associated with the use of medicines or novel therapies.

Supplementation of specific carbohydrates results in enhanced deposition of chondrogenic-specific matrix during mesenchymal stem cell differentiation.

Repair or regeneration of hyaline cartilage in knees, shoulders, intervertebral discs, and other assorted joints is a major therapeutic target. To date, therapeutic strategies utilizing chondrocytes or mesenchymal stem cells are limited by expandability or the generation of mechanically inferior cartilage. Our objective is to generate robust cartilage-specific matrix from human mesenchymal stem cells suitable for further therapeutic development. Human mesenchymal stem cells, in an alginate 3D format, were supplied with individual sugars and chains which comprise the glycan component of proteoglycans in articular cartilage (galactose, hyaluronic acid, glucuronic acid, and xylose) during chondrogenesis. After an initial evaluation for proteoglycan deposition utilizing Alcian blue, the tissue was further evaluated for viability, structural elements, and hypertrophic status. With the further addition of serum, a substantial increase was observed in viability, the amount of proteoglycan deposition, glycosaminoglycan production, and an enhancement of Hyaluronic Acid, Collagen II and Aggrecan deposition. Suppression of hypertrophic markers (COL1A1, COL10A1, MMP13, and RUNX2) was also observed. When mesenchymal stem cells were supplied with the raw building materials of proteoglycans and a limited amount of serum during chondrogenesis, it resulted in the generation of viable hyaline-like cartilage with deposition of structural components which exceeded previously reported in vitro-based cartilage.

Neural Stem Cell Tumorigenicity and Biodistribution Assessment for Phase I Clinical Trial in Parkinson's Disease.

Human pluripotent stem cells (PSC) have the potential to revolutionize regenerative medicine. However undifferentiated PSC can form tumors and strict quality control measures and safety studies must be conducted before clinical translation. Here we describe preclinical tumorigenicity and biodistribution safety studies that were required by the US Food and Drug Administration (FDA) and Australian Therapeutic Goods Administration (TGA) prior to conducting a Phase I clinical trial evaluating the safety and tolerability of human parthenogenetic stem cell derived neural stem cells ISC-hpNSC for treating Parkinson's disease (ClinicalTrials.gov Identifier NCT02452723). To mitigate the risk of having residual PSC in the final ISC-hpNSC population, we conducted sensitive in vitro assays using flow cytometry and qRT-PCR analyses and in vivo assays to determine acute toxicity, tumorigenicity and biodistribution. The results from these safety studies show the lack of residual undifferentiated PSC, negligible tumorigenic potential by ISC-hpNSC and provide additional assurance to their clinical application.

Neural Stem Cells Derived from Human Parthenogenetic Stem Cells Engraft and Promote Recovery in a Nonhuman Primate Model of Parkinson's Disease.

Cell therapy has attracted considerable interest as a promising therapeutic alternative for patients with Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying potentially viable human embryos and can be used to generate an unlimited supply of neural cells for transplantation. We have previously reported that human parthenogenetic stem cell-derived neural stem cells (hpNSCs) successfully engraft, survive long term, and increase brain dopamine (DA) levels in rodent and nonhuman primate models of PD. Here we report the results of a 12-month transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical parkinsonian symptoms. The hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immunosuppressed monkeys. Transplantation of hpNSCs was safe and well tolerated by the animals with no dyskinesia, tumors, ectopic tissue formation, or other test article-related serious adverse events. We observed that hpNSCs promoted behavioral recovery; increased striatal DA concentration, fiber innervation, and number of dopaminergic neurons; and induced the expression of genes and pathways downregulated in PD compared to vehicle control animals. These results provide further evidence for the clinical translation of hpNSCs and support the approval of the world's first pluripotent stem cell-based phase I/IIa study for the treatment of PD (Clinical Trial Identifier NCT02452723).