Modeling gonadal development and function with human pluripotent stem cells

Tutkimustuotos: OpinnäyteVäitöskirjaArtikkelikokoelma

Abstrakti

Human gonads (testes and ovaries) develop from mesoderm as paired organs and are initially identical, or bipotent, in males and females. Upon sex determination, the gonads initiate differential transcription, molecular signaling, and hormonal patterns and begin to display their sex-specific characteristics. Occasionally, these complex processes become disrupted, causing impaired gonadal development at embryonic and early fetal stages and altered gonadal functions, which may lead to infertility or even abnormal sex development. The molecular mechanisms delineating early gonadal development and sex determination are not thoroughly understood and have been challenging to investigate in humans. Human pluripotent stem cells (hPSCs), comprising human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are a versatile tool for studying regulatory mechanisms of human organ development and related disease processes. hPSCs can form all three germ layers of an early embryo and have an endless capacity to self-renew, providing an infinite source of cells equivalent to the undifferentiated cells of an embryo. Follicle-stimulating hormone (FSH) and its receptor (FSHR) are necessary for normal development and function of the reproductive system. Any disruptions in FSH-FSHR signaling may cause severe fertility issues. For example, inactivating FSHR mutation resulting in an alanine to valine (A189V) amino acid substitution leads in women to amenorrhea and infertility due to blocked follicle development and in men to markedly reduced spermatogenic capacity but partially preserved fertility. The disease mechanisms for the observed phenotypes remain obscure. In this study, the first aim was to reveal regulatory pathways and signaling mechanisms involved in early gonadal development by developing an hPSC-based in vitro model. The second aim was to evaluate the regulatory role of nuclear receptor subfamily 5 group A member 1 (NR5A1), a gene encoding steroidogenic factor 1 (SF1), in early gonadal cell transcriptomics. The third aim was to examine the function of human wild-type (WT) FSHR and FSHR with an inactivating A189V mutation. To model gonadal development, we differentiated hPSCs into bipotential gonadal-like cells via primitive streak- and intermediate mesoderm-like stages by stimulating activin, bone morphogenetic protein, and Wingless-related integration site-dependent signaling, demonstrating the importance of the proper balance between these signaling pathways in early gonadal differentiation. hiPSCs of male origin, differentiated to the bipotential gonadal-like stage, were further directed towards more mature gonadal-like anti-Müllerian hormone-producing and steroidogenic cells by activating endogenous NR5A1 with CRISPR-Cas9 technology. Cells differentiated in the absence of NR5A1 activation did not exhibit these characteristics, as assayed by gene expression and immunodetection-based methods and mass spectrometry. Moreover, by performing whole sample RNA sequencing analysis for the cells in the presence and absence of NR5A1 activation, we identified many novel targets of NR5A1 and altered gene expression patterns of many gonad-related genes and genes not previously associated with gonads in response to NR5A1. We also established a protocol for directed differentiation of hPSCs into cells endogenously expressing FSHR. By differentiating hESCs expressing WT FSHR and patient-specific hiPSCs expressing mutated A189V FSHR and by performing immunosorbent-based functional assays in the differentiated cells, we demonstrated distinct functional response to FSH between the mutated and WT receptors. In addition, we identified several novel FSHR-protein interactions by using affinity proximity mass spectrometry in HEK293 cells stably expressing WT or A189V FSHRs in stimulated and unstimulated conditions. This research project provided novel insights into signaling and gene regulatory mechanisms during early gonadal development. The established differentiation protocols can serve as promising tools also in future studies. For example, the gonadal models can be used to examine gonadal dysgenesis or causes of infertility with patient-derived hiPSCs. This project also yielded a considerable amount of publicly available transcriptomics and proteomics data, which can be utilized in evaluating SF1 and FSHR biology.
Alkuperäiskielienglanti
Valvoja/neuvonantaja
  • Tapanainen, Juha, Valvoja
  • Tuuri, Timo, Valvoja
  • Lundin (Stenroos), Karolina, Valvoja
JulkaisupaikkaHelsinki
Kustantaja
Painoksen ISBN978-951-51-8578-5
Sähköinen ISBN978-951-51-8579-2
TilaJulkaistu - 2022
OKM-julkaisutyyppiG5 Tohtorinväitöskirja (artikkeli)

Lisätietoja

M1 - 130 s. + liitteet

Tieteenalat

  • 3111 Biolääketieteet
  • 1182 Biokemia, solu- ja molekyylibiologia
  • 3123 Naisten- ja lastentaudit

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