Browsing by Subject "Naïve pluripotent stem cells"

Pluripotent stem cells (PSC) can exists in both primed and naïve states. The conventionally derived human PSCs represent the later primed state of pluripotency during embryo development, while the naïve state resembles the inner cell mass (ICM) of pre-implantation blastocyst. Primed human PSCs can be reverted chemically by transient histone deacetylase (HDAC) inhibition back to the naïve state in vitro. The reverted PSCs can then be characterized based on their morphology and expression of selected naïve markers using immunocytochemistry and RT-qPCR assays. Leucine twenty homeobox (LEUTX) is one of the genes expressed during the early stages of embryo development and is capable of activating the transcription of multiple genes, including pluripotency-associated genes, which are upregulated during the human embryonic genome activation (EGA). LEUTX expression could potentially improve the naïve reversion efficiency or the maintenance of naïve PSCs by driving the transcriptome of primed PSCs back towards the earlier cell stages of embryo development, potentially even to cell stages that precede the naïve state. The aim of this thesis was to setup the naïve reversion protocol and study the effects of LEUTX on the reversion by using the generated and tested H9 activator cell line for targeted activation of endogenous LEUTX expression. First, a conditionally stabilized CRISPRa activator cell line was generated for targeted activation of endogenous gene expression in H9 cells. Then sequence-specific guide RNAs (gRNA) targeting LEUTX for activation were introduced to the activator cell line. Using the generated activator cell line during the naïve reversions allows the targeted activation of specific genes, here LEUTX, and thus enables studying the effects of these genes on PSCs during the naïve reversion protocol. The induced activator cells expressing LEUTX managed to form four times as many naïve resembling colonies during the reversion compared to the controls, but most of these were lost after changing the medium conditions towards the end of the protocol. After the reversion was complete, the reverted PSCs were characterized as naïve PSCs based on their domed morphology and the high expression of naïve markers NANOG, KLF17, TFCP2L1 and DNMT3L when compared to the primed PSCs. The naïve reversion protocol was set up and optimized successfully and can now be used as a reliable way of obtaining human naïve PSCs for further experiments studying and modelling the earlier developmental stages during embryo development. Furthermore, the generated H9 activator cell line worked as intended and can be utilized for studying the effects of other targeted genes during the reversion or in the reverted naïve PSCs.