Shuyan Wang

Lmx1a enhances the effect of iNSCs in a PD model.

Lmx1a plays a central role in the specification of dopaminergic (DA) neurons, which potentially could be employed as a key factor for trans-differentiation to DA neurons. In our previous study, we have converted somatic cells directly into neural stem cell-like cells, namely induced neural stem cells (iNSCs), which further can be differentiated into subtypes of neurons and glia in vitro. In the present study, we continued to test whether these iNSCs have therapeutic effects when transplanted into a mouse model of Parkinsons disease (PD), especially when Lmx1a was introduced into these iNSCs under a Nestin enhancer. iNSCs that over-expressed Lmx1a (iNSC-Lmx1a) gave rise to an increased yield of dopaminergic neurons and secreted a higher level of dopamine in vitro. When transplanted into mouse models of PD, both groups of mice showed decreased ipsilateral rotations; yet mice that received iNSC-Lmx1a vs. iNSC-GFP exhibited better recovery. Although few iNSCs survived 11weeks after transplantation, the improved motor performance in iNSC-Lmx1a group did correlate with a greater tyrosine hydroxylase (TH) signal abundance in the lesioned area of striatum, suggesting that iNSCs may have worked through a non-autonomous manner to enhance the functions of remaining endogenous dopaminergic neurons in brain.

Autologous iPSC-derived dopamine neuron transplantation in a nonhuman primate Parkinson’s disease model

Autologous dopamine (DA) neurons are a new cell source for replacement therapy of Parkinson’s disease (PD). In this study, we tested the safety and efficacy of autologous induced pluripotent stem cell (iPSC)-derived DA cells for treatment of a cynomolgus monkey PD model. Monkey bone marrow mesenchymal cells were isolated and induced to iPSCs, followed by differentiation into DA cells using a method with high efficiency. Autologous DA cells were introduced into the brain of a cynomolgus monkey PD model without immunosuppression; three PD monkeys that had received no grafts served as controls. The PD monkey that had received autologous grafts experienced behavioral improvement compared with that of controls. Histological analysis revealed no overgrowth of grafts and a significant number of surviving A9 region-specific graft-derived DA neurons. The study provided a proof-of-principle to employ iPSC-derived autologous DA cells for PD treatment using a nonhuman primate PD model.

Autologous transplantation of GDNF-expressing mesenchymal stem cells protects against MPTP-induced damage in cynomolgus monkeys.

Glial cell-derived neurotrophic factor (GDNF) has shown beneficial effects in models of Parkinsons disease. The mild results observed in the double-blind clinical trial by intraputamenal infusion of recombinant GDNF proteins warrant a search for alternative delivery methods. In this study, we investigated the function of autologous mesenchymal stem cells (MSCs) expressing GDNF (GDNF-MSCs) for protection against MPTP-induced injury in cynomolgus monkeys. MSCs were obtained from the bone marrow of individual monkeys and gene-modified to express GDNF. Following unilateral engraftment of GDNF-MSCs into the striatum and substantia nigra, the animals were challenged with MPTP to induce a stable systemic Parkinsonian state. The motor functions were spared in the contralateral limbs of monkeys receiving GDNF-MSCs, but not in those receiving MSCs alone. In the striatum of the grafted hemisphere, dopamine levels were higher and dopamine uptake was enhanced. The results suggest that autologous MSCs may be a safe vehicle to deliver GDNF for enhancing nigro-striatum functions.

Advanced glycation end products and ultrastructural changes in corneas of long-term streptozotocin-induced diabetic monkeys.

Purpose: The purpose of this study was to investigate the ultrastructural corneal changes of chronic diabetic monkeys and explore the relationship between advanced glycation end products and ultrastructural changes in diabetic corneas. Methods: A total of 8 cynomolgus monkeys were used in this experiment. Four monkeys were induced into insulin-dependent diabetes mellitus for 4 years. Four age-matched healthy monkeys were used as the controls. Ultrathin sections obtained from the corneas were examined by transmission electron microscopy. Results: Advanced glycation end product immunoreactivity was observed in the epithelial cells, epithelial basement membrane, and stromal keratocytes of diabetic corneas, whereas advanced glycation end product immunoreactivity was not found in the corresponding area in normal corneas. Abnormal collagen fibril bundles of variable thickness were identified in corneal stroma in all diabetic monkeys. Epithelial and endothelial cell degeneration was also observed in 1 diabetic monkey. Conclusions: Abnormal aggregates of collagen fibrils in stromal matrix were common among long-term diabetic monkeys, and the formation of the abnormal collagen fibril aggregates might result from excessive nonenzymatic glycosylation.

Conversion of adult human peripheral blood mononuclear cells into induced neural stem cell by using episomal vectors.

Human neural stem cells (NSCs) hold great promise for research and therapy in neural diseases. Many studies have shown direct induction of NSCs from human fibroblasts, which require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Peripheral blood (PB) is routinely used in medical diagnoses, and represents a noninvasive and easily accessible source of cells. Here we show direct derivation of NSCs from adult human PB mononuclear cells (PB-MNCs) by employing episomal vectors for transgene delivery. These induced NSCs (iNSCs) can expand more than 60 passages, can exhibit NSC morphology, gene expression, differentiation potential, and self-renewing capability and can give rise to multiple functional neural subtypes and glial cells in vitro. Furthermore, the iNSCs carry a specific regional identity and have electrophysiological activity upon differentiation. Our findings provide an easily accessible approach for generating human iNSCs which will facilitate disease modeling, drug screening, and possibly regenerative medicine.

Characterizing the induction of diabetes in juvenile cynomolgus monkeys with different doses of streptozotocin

Juvenile (2–23 years old) cynomolgus monkeys are frequently used as recipients in non-human primate islet transplantation studies. The aim of this study was to examine the effects of different doses of streptozotocin (STZ), and find the optimal dose for inducing diabetes in these monkeys. Fifteen juvenile (2–3 years old) cynomolgus monkeys were separated into three groups and administered with different doses of STZ (100, 68 or 60 mg kg−1). Basal and glucose-stimulated blood glucose, insulin, and C-peptide levels, as well as body weights were monitored. Hepatic and renal function tests and pancreatic immunohistochemistry were performed before and after STZ treatment. Monkeys treated with both 100 and 68 mg kg−1 of STZ exhibited continuous hyperglycemia, which coincided with a nearly complete loss of islet β-cells. Two monkeys received 60 mg kg−1 of STZ, but only one became completely diabetic. During the first week following STZ treatment, hepatic and renal function slightly increased in these three groups. However, 24 hours post-STZ, serum total bile acid levels were significantly increased in monkeys treated with 100 mg kg−1 than those treated with 68 mg kg−1 of STZ (P<0.05). These data suggest that 100 mg kg−1 and 68 mg kg−1 of STZ can safely induce diabetes in cynomolgus monkeys aged 2–3 years, but 68 mg kg−1 of STZ, rather than 100 mg kg−1 of STZ, may be more appropriate for inducing diabetes in these monkeys. Furthermore, body surface area, rather than body weight, was a more reliable determinant of dosage, where 700 mg m−2 of STZ should be the lower limit for inducing diabetes in juvenile monkeys.

Insulin‐producing cells from human pancreatic islet‐derived progenitor cells following transplantation in mice

Stem/progenitor cells hold promise for alleviating/curing type 1 diabetes due to the capacity to differentiate into functional insulin‐producing cells. The current study aims to assess the differentiation potential of human pancreatic IPCs (islet‐derived progenitor cells). IPCs were derived from four human donors and subjected to more than 2000‐fold expansion before turning into ICCs (islet‐like cell clusters). The ICCs expressed ISL‐1 Glut2, PDX‐1, ngn3, insulin, glucagon and somatostatin at the mRNA level and stained positive for insulin and glucagon by immunofluorescence. Following glucose challenge in vitro, C‐peptide was detected in the sonicated ICCs, instead of in the conditioned medium. To examine the function of the cells in vivo, IPCs or ICCs were transplanted under the renal capsule of immunodeficient mice. One month later, 19 of 28 mice transplanted with ICCs and 4 of 14 mice with IPCs produced human C‐peptide detectable in blood, indicating that the in vivo environment further facilitated the maturation of ICCs. However, among the hormone‐positive mice, only 9 of 19 mice with ICCs and two of four mice with IPCs were able to secrete C‐peptide in response to glucose.

Inducible regulation of GDNF expression in human neural stem cells

Glial cell derived neurotrophic factor (GDNF) holds promises for treating neurodegenerative diseases such as Parkinson’s disease. Human neural stem cells (hNSCs) have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin resistance gene as such vehicles. A modified tetracycline operator 7 (tetO7) was inserted into a region upstream of the EF1-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein (TTS). TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline (Dox) the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-1 positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.

Accelerated generation of oligodendrocyte progenitor cells from human induced pluripotent stem cells by forced expression of Sox10 and Olig2

Oligodendrocyte progenitor cells (OPCs) hold great promise for treatment of dysmyelinating disorders, such as multiple sclerosis and cerebral palsy. Recent studies on generation of human OPCs mainly use human embryonic stem cells (hESCs) or neural stem cells (NSCs) as starter cell sources for the differentiation process. However, NSCs are restricted in availability and the present method for generation of oligodendrocytes (OLs) from ESCs often requires a lengthy period of time. Here, we demonstrated a protocol to efficiently derive OPCs from human induced pluripotent stem cells (hiPSCs) by forced expression of two transcription factors (2TFs), Sox10 and Olig2. With this method, PDGFRα+ OPCs can be obtained in 14 days and O4+ OPCs in 56 days. Furthermore, OPCs may be able to differentiate to mature OLs that could ensheath axons when co-cultured with rat cortical neurons. The results have implications in the development of autologous cell therapies.

SIRT6 deficiency results in developmental retardation in cynomolgus monkeys

SIRT6 acts as a longevity protein in rodents1,2. However, its biological function in primates remains largely unknown. Here we generate a SIRT6-null cynomolgus monkey (Macaca fascicularis) model using a CRISPR–Cas9-based approach. SIRT6-deficient monkeys die hours after birth and exhibit severe prenatal developmental retardation. SIRT6 loss delays neuronal differentiation by transcriptionally activating the long non-coding RNA H19 (a developmental repressor), and we were able to recapitulate this process in a human neural progenitor cell differentiation system. SIRT6 deficiency results in histone hyperacetylation at the imprinting control region of H19, CTCF recruitment and upregulation of H19. Our results suggest that SIRT6 is involved in regulating development in non-human primates, and may provide mechanistic insight into human perinatal lethality syndrome.A cynomolgus monkey model deficient in SIRT6 protein exhibits severe retardation in prenatal development, in which neuronal differentiation is delayed by activation of the H19 long non-coding RNA.