Generating growth hormone-releasing hormone neurons from human embryonic stem cells

Informations projet

GHRH neurons

N° de convention de subvention: 894596

DOI

10.3030/894596

Projet clôturé

Date de signature de la CE 12 Mai 2020

Date de début 1 Septembre 2020

Date de fin 31 Août 2022

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 202 680,96

Contribution de l’UE

€ 202 680,96

Coordonné par

HELSINGIN YLIOPISTO
Finland

Periodic Reporting for period 1 - GHRH neurons (Generating growth hormone-releasing hormone neurons from human embryonic stem cells)

Période du rapport: 2020-09-01 au 2022-08-31

Résumé du contexte et des objectifs généraux du projet

The objective of this project is to generate growth hormone-releasing hormone (GHRH) neurons from human embryonic stem cells (hESCs) through both conventional growth factor-based method and CRISPR-Cas9-based gene activation (CRISPRa) direct programming method. This will provide a platform for disease modeling (growth hormone deficiency), allow drug screening for growth disorders, and renew science by using new technology to answer clinically relevant questions.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

1 Work Package 1
-Results
a. I tested existed protocols to generate hypothalamic neurons from human embryonic stem cells, and found that only our in-house GnRH neuron differentiation protocol can induce GHRH expression to some extent.
b. Combined with current understanding about GHRH neuron/hypothalamus development, I included SHH signaling activator and Wnt signaling inhibitor in the differentiation, and found they can further increase the GHRH expression.
c. I established the GHRH-tdTomato hESC line to report the GHRH expression level, which will facilitate the differentiation protocol optimization.
d. I collaborated with FIMM High Content Imaging and Analysis (FIMM-HCA) unit and performed a chemical library screen with the established GHRH-tdTomato hESC line. It was shown that EZH2 inhibitor could enhance the tdTomato signal significantly. So I further optimized the duration and concentration of EZH2 inhibitor to get efficient GHRH induction.
e. I have performed the single cell RNA sequencing (scRNA-seq) with our fully established GnRH neuron differentiation protocol to get trained with the sample preparation and data analysis for scRNA-seq.
2 Work Package 2
-Results
a. I generated DDdCas9-VP192 GHRH-tdTomato hESC line, which could report the GHRH expression level as well as expressing transcription activator connected with dCas9.
b. I established the vector containing guide RNAs targeting the promotors of ASCL1 and GHRH.
c. I could directly programed the hESCs into GHRH positive cells with ASCL1 and GHRH transcriptional activation.
3 Work Package 3
-Results
a. I introduced KCNQ1 mutation (R116L) in hESCs with CRISPR/Cas9 technique.
b. I tested the differentiation protocol developed from work package 1 in both wild-type hESC line and R116L mutation hESC line. However there is no difference in GHRH neuron induction. This indicates that the downstream of GHRH could be impaired in the KCNQ1 mutation patient with growth hormone deficiency.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

To the best of my knowledge, this is the first attempt to specifically differentiate human GHRH neurons from hESCs.

I established a conventional, well-defined protocol for growth factor-based cellular differentiation for this cell type, and also developed a method to efficiently drive their formation by direct programming with CRISPRa. Although these differentiated cells needs to be characterized in a further step, such as their intracellular packing of GHRH, the secretion of GHRH, intracellular calcium signal, electrophysiology and even in vivo functions.

These novel cell models can be used for drug-screening and development of innovative approaches to modulate endogenous growth hormone (GH) secretion, and the results should be applicable not only to patients with growth hormone deficiency (GHD) but also to those with GH excess (i.e. acromegaly). Secondly, the best current estimate for the percentage of patients that can obtain molecular genetic diagnosis for combined pituitary hormone deficiency is as low as 8%, which reflects the urgent need for better understanding of molecular genetic etiology of diseases affecting the hypothalamus. Indeed, this figure is surprisingly low in the era of next generation sequencing. Finally, height and weight have culture-bound dimensions in different countries, which is why elucidating the basic mechanisms for their regulation is expected to arouse the interest of not only stem cell researchers but also the general public.

https://academic.oup.com/stmcls/advance-article/doi/10.1093/stmcls/sxac069/6714007