Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression

Smela, Merrick Pierson; Kramme, Christian; Fortuna, Patrick R.J.; Adams, Jessica; Dong, Edward; Kobayashi, Mutsumi; Brixi, Garyk; Tysinger, Emma; Kohman, Richie E.; Shioda, Toshi; Chatterjee, Pranam; Church, George M.

doi:10.1101/2022.07.04.498717

Cited by 6 publications

(9 citation statements)

References 44 publications

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“…Contrary to other approaches, combinatorial barcoding does not require any complex microfluidics or other specialized equipment. Over the past few years, this approach has been widely adopted to study developmental processes and disease progression at scale [13][14][15][16][17][18][19][20][21][22][23][24][25][26] .…”

Section: Introductionmentioning

confidence: 99%

High sensitivity single cell RNA sequencing with split pool barcoding

Tran¹,

Papalexi²,

Schroeder³

et al. 2022

Preprint

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Single cell RNA sequencing (scRNA-seq) has become a core tool for researchers to understand biology. As scRNA-seq has become more ubiquitous, many applications demand higher scalability and sensitivity. Split-pool combinatorial barcoding makes it possible to scale projects to hundreds of samples and millions of cells, overcoming limitations of previous droplet based technologies. However, there is still a need for increased sensitivity for both droplet and combinatorial barcoding based scRNA-seq technologies. To meet this need, here we introduce an updated combinatorial barcoding method for scRNA-seq with dramatically improved sensitivity. To assess performance, we profile a variety of sample types, including cell lines, human peripheral blood mononuclear cells (PBMCs), mouse brain nuclei, and mouse liver nuclei. When compared to the previously best performing approach, we find up to a 2.6-fold increase in unique transcripts detected per cell and up to a 1.8-fold increase in genes detected per cell. These improvements to transcript and gene detection increase the resolution of the resulting data, making it easier to distinguish cell types and states in heterogeneous samples. Split-pool combinatorial barcoding already enables scaling to millions of cells, the ability to perform scRNA-seq on previously fixed and frozen samples, and access to scRNA-seq without the need to purchase specialized lab equipment. Our hope is that by combining these previous advantages with the dramatic improvements to sensitivity presented here, we will elevate the standards and capabilities of scRNA-seq for the broader community.

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Section: Introductionmentioning

confidence: 99%

High sensitivity single cell RNA sequencing with split pool barcoding

Tran¹,

Papalexi²,

Schroeder³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…For examples, overexpression of DAZL , a late germ cells marker, and BOULE , a germ cell meiotic marker, in hESCs produces DDX4- (late germ cell marker) and SYCP3- (germ meiotic marker) double positive cells, which form follicle-like structures when culture with GDF9 and BMP15 ( Jung et al, 2017 ). A recent study also used transcriptional factor library to screen critical factors for deriving granulosa cells from hiPSCs, and NR5A1 , RNUX1 or RUNX2 were identified as critical factors in granulosa cell fate decision ( Smela et al, 2022 ). The differentiated granulosa cell-like cells expressed granulosa cell markers including AMHR2 , CD82 , FOXL2 , FSHR , IGFBP7 , KRT19 , STAR , and WNT4 , and responded to exogenous FSH by producing oestrogen and progesterone ( Smela et al, 2022 ).…”

Section: Current Progress On In-vitro Differentiat...mentioning

confidence: 99%

“…A recent study also used transcriptional factor library to screen critical factors for deriving granulosa cells from hiPSCs, and NR5A1 , RNUX1 or RUNX2 were identified as critical factors in granulosa cell fate decision ( Smela et al, 2022 ). The differentiated granulosa cell-like cells expressed granulosa cell markers including AMHR2 , CD82 , FOXL2 , FSHR , IGFBP7 , KRT19 , STAR , and WNT4 , and responded to exogenous FSH by producing oestrogen and progesterone ( Smela et al, 2022 ). However, the developmental stage of the in-vitro differentiated pre-GCs is unknown ( Jung et al, 2017 ).…”

Section: Current Progress On In-vitro Differentiat...mentioning

confidence: 99%

Current progress on in vitro differentiation of ovarian follicles from pluripotent stem cells

Chen

Yeung

et al. 2023

Front. Cell Dev. Biol.

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Mammalian female reproduction requires a functional ovary. Competence of the ovary is determined by the quality of its basic unit–ovarian follicles. A normal follicle consists of an oocyte enclosed within ovarian follicular cells. In humans and mice, the ovarian follicles are formed at the foetal and the early neonatal stage respectively, and their renewal at the adult stage is controversial. Extensive research emerges recently to produce ovarian follicles in-vitro from different species. Previous reports demonstrated the differentiation of mouse and human pluripotent stem cells into germline cells, termed primordial germ cell-like cells (PGCLCs). The germ cell-specific gene expressions and epigenetic features including global DNA demethylation and histone modifications of the pluripotent stem cells-derived PGCLCs were extensively characterized. The PGCLCs hold potential for forming ovarian follicles or organoids upon cocultured with ovarian somatic cells. Intriguingly, the oocytes isolated from the organoids could be fertilized in-vitro. Based on the knowledge of in-vivo derived pre-granulosa cells, the generation of these cells from pluripotent stem cells termed foetal ovarian somatic cell-like cells was also reported recently. Despite successful in-vitro folliculogenesis from pluripotent stem cells, the efficiency remains low, mainly due to the lack of information on the interaction between PGCLCs and pre-granulosa cells. The establishment of in-vitro pluripotent stem cell-based models paves the way for understanding the critical signalling pathways and molecules during folliculogenesis. This article aims to review the developmental events during in-vivo follicular development and discuss the current progress of generation of PGCLCs, pre-granulosa and theca cells in-vitro.

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“…This represents a big challenge for the isolation of autologous follicular somatic cells to create artificial follicles. An alternative approach could be to differentiate iPSCs from menopausal individuals into granulosa and theca cells in vitro and to subsequently generate transplantable ovarian organoids, but differentiation protocols to obtain both types of somatic ovarian cells in humans are still under development and hence not yet available for clinical applications [72].…”

Section: Hormonal Production and Menopausementioning

confidence: 99%

Bioengineered 3D Ovarian Models as Paramount Technology for Female Health Management and Reproduction

Valle

Lopes

2023

Bioengineering

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Ovarian dysfunction poses significant threats to the health of female individuals. Ovarian failure can lead to infertility due to the lack or inefficient production of fertilizable eggs. In addition, the ovary produces hormones, such as estrogen and progesterone, that play crucial roles not only during pregnancy, but also in maintaining cardiovascular, bone, and cognitive health. Decline in estrogen and progesterone production due to ovarian dysfunction can result in menopausal-associated syndromes and lead to conditions, such as osteoporosis, cardiovascular disease, and Alzheimer’s disease. Recent advances in the design of bioengineered three-dimensional (3D) ovarian models, such as ovarian organoids or artificial ovaries, have made it possible to mimic aspects of the cellular heterogeneity and functional characteristics of the ovary in vitro. These novel technologies are emerging as valuable tools for studying ovarian physiology and pathology and may provide alternatives for fertility preservation. Moreover, they may have the potential to restore aspects of ovarian function, improving the quality of life of the (aging) female population. This review focuses on the state of the art of 3D ovarian platforms, including the latest advances modeling female reproduction, female physiology, ovarian cancer, and drug screening.

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Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression

Cited by 6 publications

References 44 publications

High sensitivity single cell RNA sequencing with split pool barcoding

High sensitivity single cell RNA sequencing with split pool barcoding

Current progress on in vitro differentiation of ovarian follicles from pluripotent stem cells

Bioengineered 3D Ovarian Models as Paramount Technology for Female Health Management and Reproduction

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