CAG repeat instability in embryonic stem cells and derivative spermatogenic cells of transgenic Huntington's disease monkey

Abstract

Purpose: The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington's disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time.

Methods: HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis.

Results: Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type-specific and size-dependent.

Conclusions: Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.

Keywords: CAG repeat instability; Genetic anticipation; HD rhesus monkey embryonic stem cells (rESCs); Huntington’s disease; Spermatogenic cells.

Fig. 1

Experimental flow chart. Seven rESC lines (rESCs1–7; F1) [48] inherited mutant HTT transgenes from rHD1 (F0) [49] were established from rHD1 sperm (F1) derived blastocysts (F1). rES5 and rES7 were differentiated into F2 spermatogenic cells (SCs) in vitro. M: male; F: female, WT: wild type; rES: HD monkey embryonic stem cells; rSCs: HD monkey spermatogenic cells; N/A: not applied; Y: yes; N: no

Fig. 2

CAG repeat instability in rHD1 lymphocytes and sperm. a Curve fit analysis of mutant HTT alleles. The cluster of CAG repeats are classified into allele clusters (1) <35Q, (2) 40–60Q, and (3) >60Q. Black lines represent the overall curve fit overlay onto the spectrum of each allele clusters. Red lines represent individual curves that fit to the expansion of mutant HTT alleles. The number of CAG repeats (Q) was denoted at the X-axis and by the arrows. b Distribution of CAG repeat size in lymphocytes at three and 44 months of age. The black horizontal line represents the median of each data set based on curve fit data on allele clusters of >60Q in (a). c CAG repeat mosaicism in sperm of rHD1 monkey based on curve fit data in (a)

Fig. 3

Assessments of CAG repeat instability in the long-term culture of rESs1–7. a Curve fit analysis of CAG repeat expansion in rESs1–7 at the start and the end of in vitro culture. b The average number of repeats added in allele clusters of <35Q and >60Q in rESs1–7 after long-term culture. c Expansion of CAG repeats in allele cluster of >60Q. An open and solid circle represents CAG repeat size from an individual red curve in (a) in vitro culture beginning and end, respectively. A black horizontal line represents the median of each data set. **p <0.01

Fig. 4

In vitro spermatogenic cell differentiation of rES5 and rES7. a Experimental timeline of spermatogenic cell differentiation. b-d Expression of spermatogenic cell markers on day 0 (before spermatogenic cell differentiation) and day 10 (after 10-day spermatogenic cell differentiation) of spermatogenic cell differentiation of rSC-WT (b), rSC5 (c), and rSC7 (d) by quantitative real-time PCR (qRT-PCR). *p<0.05 and n=3

Fig. 5

Assessments of CAG repeat instability in in vitro derived spermatogenic cells. a and b Curve fit analysis of rES5 and rES7 spermatogenic cell differentiation. c An average number of repeats added in allele clusters of <35Q and >60Q in spermatogenic cells derived from rES5 and rES7. d Expansion of CAG repeats in allele clusters of >60Q in spermatogenic cells derived from rES5 and rES7. The open circle represents CAG repeat size from the individual red curve in “a” at day 0 before differentiation. The solid circle represents the CAG repeat size at day10 of spermatogenic cell differentiation. The black line represents the median of each data set. *p <0.05

Fig. 6

CAG repeat instability and expansion rates in rESs1–7 and spermatogenic cells derived from rES5 and rES7. a An average number of repeats added in mutant HTT alleles of <60Q and >60Q clusters between rESCs and spermatogenic cells derived from rES5 and rES7 was presented. b Daily expansion rates of long-term cultured rESs1–7, long-term cultured rES5 and 7, and in vitro derived spermatogenic cells (rSC5 and 7). c Analysis between CAG repeat size and daily expansion rates of rESs1–7 (p = 0.3737, open circles) and in vitro derived spermatogenic cells (p = 0.0054, solid squares). *p <0.05, **p <0.01, ***p<0.001