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

Abstract

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 Huntington's disease. Additionally, a Huntington's cell model that develops robust pathological features of Huntington's disease would be valuable for drug discovery research.ResultsTo test this hypothesis, a pluripotent Huntington's disease monkey hybrid cell line (TrES1) was established from a tetraploid Huntington's disease monkey blastocyst generated by the fusion of transgenic Huntington's monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntington's 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 Huntington's disease cellular features is influenced by neural developmental events.ConclusionsHuntington's 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 Huntington's disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntington's disease pathogenesis.

Pluripotent Stem Cells to Model and Treat Huntington’s Disease

“…These cells preserve both the HD-related genotype and phenotype: they express mutant HTT protein and show formation of intracellular HTT protein aggregates [75,89,90]. In addition, PSCs have been generated using cell fusion as a tool for reprogramming: transgenic HD monkey skin fibroblasts and wild-type non-transgenic monkey oocytes were fused and the pluripotent hybrid cells selected after fusion were found to express mutant Htt and to have HTT protein intracellular inclusions after the induction of in vitro neural differentiation [91]. These studies teach us that HD pluripotent cells can recapitulate the genotype and cellular phenotype of HD-patient cells, which is crucial for the production of cell systems that closely resemble HD.…”

Pathogenic Cellular Phenotypes are Germline Transmissible in a Transgenic Primate Model of Huntington's Disease

“…Cytogenetic analysis/G-banding analysis [24,25] rHD-ESCs were treated with KaryoMax Ò colcemid (Invitrogen) for 20 min, dislodged with 0.05% Trypsin-EDTA, centrifuged, and resuspended in hypotonic 0.075 M KCl solution for 20 min. After centrifugation, the cells were fixed thrice in a 3:1 ratio of methanol to glacial acetic acid.…”

“…Immunostaining of mutant HTT [24,25] Differentiated rHD-ESCs were fixed using 4% PFA for 15 min, permeabilized, and blocked. The sample was then incubated with primary antibody mEM48 (1:50) at 4°C overnight.…”

“…Immunostaining of stem cell markers [24,25] rHD-ESC culture was fixed in 4% paraformaldehyde (PFA), permeabilized by 1% Triton-X (excluded for cell surface markers), blocked with 2% BSA and 130 mM glycine in a phosphate buffer saline (PBS). After overnight incubation with primary antibodies, Nanog (Santa Cruz Biotechnology Cat# sc-30329 Conc: 1:200), Oct4 (Santa Cruz Biotechnology Cat# sc-5279 Conc: 1:500), SSEA-4 (Chemicon Cat# MB4303 Conc: 1:250), TRA-1-60 (Chemicon MAB4360 1:200) followed by thorough washes, a secondary antibody conjugated with Alexa Red (Molecular Probe Cat# A21203 Conc: 1:1000) was used for detection of the primary antibodies.…”

“…Production of transgenic HD monkey embryos and establishment of ESCs [21,[23][24][25] MII arrested oocytes were retrieved from hormonestimulated female rhesus macaques and used for in vitro fertilization by intracytoplasmic sperm injection (ICSI) followed by in vitro culture using HECM-9 [24]. HD and WT-Control monkeys were chair trained for semen collection using IACUC approved electroejaculation protocol [20,21,23].…”