Pei-Hsun Cheng

Towards a transgenic model of Huntington’s disease in a non-human primate

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10–15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.

Putative Dental Pulp-Derived Stem/Stromal Cells Promote Proliferation and Differentiation of Endogenous Neural Cells in the Hippocampus of Mice

Until now, interest in dental pulp stem/stromal cell (DPSC) research has centered on mineralization and tooth repair. Beginning a new paradigm in DPSC research, we grafted undifferentiated, untreated DPSCs into the hippocampus of immune‐suppressed mice. The rhesus DPSC (rDPSC) line used was established from the dental pulp of rhesus macaques and found to be similar to human bone marrow/mesenchymal stem cells, which express Nanog, Rex‐1, Oct‐4, and various cell surface antigens, and have multipotent differentiation capability. Implantation of rDPSCs into the hippocampus of mice stimulated proliferation of endogenous neural cells and resulted in the recruitment of pre‐existing Nestin+ neural progenitor cells (NPCs) and β‐tubulin‐III+ mature neurons to the site of the graft. Additionally, many cells born during the first 7 days after implantation proliferated, forming NPCs and neurons, and, to a lesser extent, underwent astrogliosis, forming astrocytes and microglia, by 30 days after implantation. Although the DPSC graft itself was short term, it had long‐term effects by promoting growth factor signaling. Implantation of DPSCs enhanced the expression of ciliary neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor for up to 30 days after implantation. In conclusion, grafting rDPSCs promotes proliferation, cell recruitment, and maturation of endogenous stem/progenitor cells by modulating the local microenvironment. Our results suggest that DPSCs have a valuable, unique therapeutic potential, specifically as a stimulator and modulator of the local repair response in the central nervous system. DPSCs would be a preferable cell source for therapy due to the possibility of a “personalized” stem cell, avoiding the problems associated with host immune rejection.

Reprogramming Huntington Monkey Skin Cells into Pluripotent Stem Cells

Induced pluripotent Huntingtons disease monkey stem cells (rHD-iPSCs) were established by the overexpression of rhesus macaque transcription factors (Oct4, Sox2, and Klf4) in transgenic Huntingtons monkey skin fibroblasts. The rHD-iPSCs were pluripotent and capable of differentiating into neuronal cell types in vitro and developed teratoma in immune compromised mice. We also demonstrated the upregulation of endogenous Oct4 and Sox2 after successful reprogramming to pluripotency in rHD-iPSCs, which was not expressed in skin fibroblasts. rHD-iPSCs also developed cellular features comparable to Huntingtons disease (HD), including the accumulation of mutant huntingtin (htt) aggregate and the formation of intranuclear inclusions (NIs) paralleling neural differentiation in vitro. Induced pluripotent stem cells from transgenic HD monkeys open a new era of nonhuman primate modeling of human diseases. rHD-iPSCs that develop key HD cellular features and parallel neural differentiation can be a powerful platform for investigating the developmental impact on HD pathogenesis and developing new therapies, which can be evaluated in HD monkeys from whom the rHD-iPSCs were derived.

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

BackgroundBackgroundChimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.ResultsChDPSCs were isolated from an incisor and a canine of a forty-seven year old female chimpanzee. A homogenous population of ChDPSCs was established in early culture at a high proliferation rate and verified by the expression pattern of thirteen cell surface markers. The ChDPSCs are multipotent and were capable of differentiating into osteogenic, adipogenic and chondrogenic lineages under appropriate in vitro culture conditions. ChDPSCs also express stem cell (Sox-2, Nanog, Rex-1, Oct-4) and osteogenic (Osteonectin, osteocalcin, osteopontin) markers, which is comparable to reported results of rhesus monkey BMSCs (rBMSCs), hBMSCs and hDPSCs. Although ChDPSCs vigorously proliferated during the initial phase and gradually decreased in subsequent passages, the telomere length indicated that telomerase activity was not significantly reduced.ConclusionThese results demonstrate that ChDPSCs can be efficiently isolated from post-mortem teeth of adult chimpanzees and are multipotent. Due to the almost identical genome composition of humans and chimpanzees, there is an emergent need for defining the new role of chimpanzee modeling in comparative medicine. Teeth are easy to recover at necropsy and easy to preserve prior to the retrieval of dental pulp for stem/stromal cells isolation. Therefore, the establishment of ChDPSCs would preserve and maximize the applications of such a unique and invaluable animal model, and could advance the understanding of cellular functions and differentiation control of adult stem cells in higher primates.

Longitudinal monitoring of stem cell grafts in vivo using magnetic resonance imaging with inducible maga as a genetic reporter.

Purpose: The ability to longitudinally monitor cell grafts and assess their condition is critical for the clinical translation of stem cell therapy in regenerative medicine. Developing an inducible genetic magnetic resonance imaging (MRI) reporter will enable non-invasive and longitudinal monitoring of stem cell grafts in vivo. Methods: MagA, a bacterial gene involved in the formation of iron oxide nanocrystals, was genetically modified for in vivo monitoring of cell grafts by MRI. Inducible expression of MagA was regulated by a Tet-On (Tet) switch. A mouse embryonic stem cell-line carrying Tet-MagA (mESC-MagA) was established by lentivirus transduction. The impact of expressing MagA in mESCs was evaluated via proliferation assay, cytotoxicity assay, teratoma formation, MRI, and inductively coupled plasma atomic emission spectroscopy (ICP-OES). Mice were grafted with mESCs with and without MagA (mESC-MagA and mESC-WT). The condition of cell grafts with induced “ON” and non-induced “OFF” expression of MagA was longitudinally monitored in vivo using a 7T MRI scanner. After imaging, whole brain samples were harvested for histological assessment. Results: Expression of MagA in mESCs resulted in significant changes in the transverse relaxation rate (R2 or 1/T2) and susceptibility weighted MRI contrast. The pluripotency of mESCs carrying MagA was not affected in vitro or in vivo. Intracranial mESC-MagA grafts generated sufficient T2 and susceptibility weighted contrast at 7T. The mESC-MagA grafts can be monitored by MRI longitudinally upon induced expression of MagA by administering doxycycline (Dox) via diet. Conclusion: Our results demonstrate MagA could be used to monitor cell grafts noninvasively, longitudinally, and repetitively, enabling the assessment of cell graft conditions in vivo.

Lentiviral integration preferences in transgenic mice.

Lentiviral gene transfer has a significant impact on the development of biomedical research. One of the most important features of lentiviruses is the capability to infect both dividing and nondividing cells. However, little is known whether integration preference exists, specifically in early embryos. An in‐depth genome analysis on 112 independent lentiviral integration sites from 43 transgenic founder mice was performed to determine if there are preferable sites for lentiviral integration in early embryonic genome. Our results demonstrated that lentiviruses were biased in integrating within intragenic regions, especially in the introns. However, no integration preference was found associated with specific chromosomes, repetitive elements, or CpG islands, nor was there any preference for integrating at close proximity to transcription start sites. Our findings suggested that lentiviruses were biased to integrate into the intragenic regions of early embryonic genome of mouse. genesis 46:711–718, 2008.

Monkey hybrid stem cells develop cellular features of Huntington's disease.

BackgroundPluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntingtons disease. Additionally, a Huntingtons cell model that develops robust pathological features of Huntingtons disease would be valuable for drug discovery research.ResultsTo test this hypothesis, a pluripotent Huntingtons disease monkey hybrid cell line (TrES1) was established from a tetraploid Huntingtons disease monkey blastocyst generated by the fusion of transgenic Huntingtons monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntingtons disease cellular pathological features that paralleled neural development. It expressed mutant huntingtin and stem cell markers, was capable of differentiating to neural cells, and developed teratoma in severely compromised immune deficient (SCID) mice. Interestingly, the expression of mutant htt, the accumulation of oligomeric mutant htt and the formation of intranuclear inclusions paralleled neural development in vitro , and even mutant htt was ubiquitously expressed. This suggests the development of Huntingtons disease cellular features is influenced by neural developmental events.ConclusionsHuntingtons disease cellular features is influenced by neural developmental events. These results are the first to demonstrate that a pluripotent stem cell line is able to mimic Huntingtons disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntingtons disease pathogenesis.

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

BackgroundDental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntingtons disease (HD), have not been fully investigated.ResultsTo examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile.ConclusionsOur results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.

Fibroblast Growth Factor 9 Suppresses Striatal Cell Death Dominantly Through ERK Signaling in Huntington’s Disease

Background/Aims: Huntington’s disease (HD) is a heritable neurodegenerative disorder, and there is no cure for HD to date. A type of fibroblast growth factor (FGF), FGF9, has been reported to play prosurvival roles in other neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. However, the effects of FGF9 on HD is still unknown. With many similarities in the cellular and pathological mechanisms that eventually cause cell death in neurodegenerative diseases, we hypothesize that FGF9 might provide neuroprotective functions in HD. Methods: In this study, STHdhQ7/Q7 (WT) and STHdhQ111/Q111 (HD) striatal knock-in cell lines were used to evaluate the neuroprotective effects of FGF9. Cell proliferation, cell death and neuroprotective markers were determined via the MTT assay, propidium iodide staining and Western blotting, respectively. The signaling pathways regulated by FGF9 were demonstrated using Western blotting. Additionally, HD transgenic mouse models were used to further confirm the neuroprotective effects of FGF9 via ELISA, Western blotting and immunostaining. Results: Results show that FGF9 not only enhances cell proliferation, but also alleviates cell death as cells under starvation stress. In addition, FGF9 significantly upregulates glial cell line-derived neurotrophic factor (GDNF) and an anti-apoptotic marker, Bcl-xL, and decreases the expression level of an apoptotic marker, cleaved caspase 3. Furthermore, FGF9 functions through ERK, AKT and JNK pathways. Especially, ERK pathway plays a critical role to influence the effects of FGF9 toward cell survival and GDNF production. Conclusions: These results not only show the neuroprotective effects of FGF9, but also clarify the critical mechanisms in HD cells, further providing an insight for the therapeutic potential of FGF9 in HD.