Studying The Effect Of The Treatment Using HiPSC-Derived MSN On Huntington’s Disease In Rats

Objective: To establish an efficient,rapid and safe in vitro experimental protocol for differentiation of hiPSC into MSN;and to implant the obtained MSN into the striatum of HD rats for verifying the effect of treatment.Methods:(1)Establish a set of in vitro culture program for hiPSC to be induced and differentiated to MSN and verify obtained MSN to meet the yield and quality requirements in this study.(2)Detect MSN development at each stage of the induced differentiation process by real-time quantitative PCR and immunofluorescence staining to verify the effect of the induction culture;using NGS(the second generation sequencing)to detect the expression of transcription products at different stages of the induced differentiation,such as OCT4,DLX5,GAD6 and DARPP32 and other genes,and compared the expression levels of these genes in MSN with the expression levels in undifferentiated hiPSC.(3)Use quinolinic acid(QA)to model rats to show symptoms similar to HD and evaluate the injury degree of the model.(4)The produced MSN was implanted into the injury area of HD rat brain,and the effect of the transplantation was detected by HE staining and immunofluorescence staining;(5)41 SD rats were randomly divided into four groups: sham group(n=10),vehicle group(n=10),QA+HBSS group(n=10),and QA+MSN group(n=11),among which the QA+MSN group was microinjected of MSN,and QA+HBSS group was given HBSS injection.On day 28 th after cell or HBSS transplantation,rats were sacrificed,and their brains were taken to observe for examination of their brain damage and cell survival.(6)All rats in the above4 groups were tested on the neurological behavior scale and the rotating rod test 2 days after QA modeling,on day 1,day 7,day 14,day 21 and day 28 after the operation.Results:(1)Comparing to undifferentiated hiPSC,MSN transcripts can express its specific markers GAD67 and DARPP32 at a higher level(P<0.0001),and MSNP transcripts can express its specific markers DLX5 gene at high level of expression(P<0.001),while both populations have relatively low expression of OCT4,a specific marker of undifferentiated hiPSC(P<0.0001).(2)QA modeling triggered changes in brain tissue structure,brain striatum damage and the appearance of HD neurological and behavioral symptoms in rats.(3)The results of immunofluorescence staining show that MSN can be successfully implanted into the HD rat’s striatum,and MSN can survive in the striatum for a longer period of time after implantation;(4)Comparing to those control rats without MSN treatment,rats implanted with MSN have better performance in sensory and motor abilities,and their HD-like symptoms improved significantly over time four weeks after MSN transplantation(P<0.01).Conclusion:(1)In vitro,hiPSC can be used as a starting point to achieve highefficient and rapid induction of MSN with high quality.(2)It is relatively safe to use MSN with a higher degree of differentiation for cell transplantation,which can ensure lower tumorigenicity and better transplantation effect.(3)The transplantation of MSN into the HD rat striatum can improve their symptoms of HD and restore certain neurobehavioral performance in HD rats.