Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers

Kobayashi, Mutsumi; Kobayashi, Misato; Odajima, Junko; Shioda, Keiko; Hwang, Young Sun; Sasaki, Kotaro; Chatterjee, Pranam; Kramme, Christian; Kohman, Richie E.; Church, George M.; Loehr, Amanda R.; Weiss, Robert S.; Jüppner, Harald; Gell, Joanna J.; Lau, Ching C.; Shioda, Toshi

doi:10.1016/j.stemcr.2022.01.012

Cited by 13 publications

(43 citation statements)

References 29 publications

(78 reference statements)

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“…By immunofluorescence, we observed expression of the mature marker DAZL beginning in a subset of OCT4+ hPGCLCs at 4 days of culture (Figure 5A). This is notable in comparison to a previous study using the same hPGCLC line and anti-DAZL antibody, in which DAZL staining was observed only after 77 days of co-culture with mouse fetal testis somatic cells 34 . Expression of TFAP2C, an early-stage PGC marker, declined during ovaroid culture (Figure 5B) and was almost entirely absent by day 8.…”

Section: Granulosa-like Cells Support Germ Cell Maturation Within Ova...contrasting

confidence: 51%

“…Our system has several advantages in comparison with existing in vitro models of the human ovary. Several studies have used mouse gonadal cells to support hPGCLC maturation 2,34 , but this process is much slower (~77 days vs. ~4 days in our protocol) and the mouse cells may not behave the same as human cells. Other studies have reported growth-factor based protocols to differentiate human pluripotent stem cells to ovarian granulosa-like cells [8][9][10] .…”

Section: Discussionmentioning

confidence: 99%

“…Passaging was performed with TRYPLE (Gibco). hPGCLCs were cultured in S-CM medium as previously described 34 , and passaged with Accutase (Stemcell Technologies). Mycoplasma testing was performed by PCR every 3 months; all cells tested negative.…”

Section: Cell Culturementioning

confidence: 99%

“…Ovaroid formation with hPGCLCs and granulosa-like cells F2 female hPGCLCs were maintained in long-term culture medium as previously described 34 and harvested with Accutase. To form ovaroids, granulosa-like cells were harvested with TRYPLE, counted, and mixed with F2 hPGCLCs.…”

Section: Rna-seqmentioning

confidence: 99%

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

Smela

Kramme

Fortuna

et al. 2022

Preprint

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An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells form ovary-like organoids (ovaroids) when aggregated with hPGCLCs, and recapitulate key ovarian phenotypes including support of germ cell maturation, follicle formation, and steroidogenesis. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

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Section: Granulosa-like Cells Support Germ Cell Maturation Within Ova...contrasting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Rna-seqmentioning

confidence: 99%

See 2 more Smart Citations

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

Smela

Kramme

Fortuna

et al. 2022

Preprint

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“…Serendipitously, we find that by seeding isolated iOLCs onto matrigel-coated plates feeder-free in a media composition recently described for hPGCLC expansion, we are able to maintain and expand the iOLC population (Figure S7A). 12 iOLCs are maintained in culture for over 30 days, retaining heterogeneous expression of DDX4. These DDX4+ iOLCs are larger than DDX4-cells of hiPSCs, averaging 100 μm in width and growing in nest-like structures (Figure S7A and S7B).…”

Section: Iolcs Expand Feeder-free In Culturementioning

confidence: 99%

Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors

Kramme

Smela

Wolf

et al. 2022

Preprint

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Germ cells are the vehicle of human reproduction, arising early in embryonic development and developing throughout adult life until menopause onset in women. Primordial germ cells are the common precursors of germline cells in both sexes, undergoing sexual specification into oogonia or gonocytes which further develop into oocytes or spermatocytes during development. Methods for recapitulation of primordial germ cell and oogonia formation have been developed extensively in recent decades, but fundamental technical limitations in their methodologies, throughput, and yield limit their utilization. Recently, transcription factor (TF)-based methods for human primordial germ cell-like cell (hPGCLC) formation, mouse meiotic entry, and mouse oocyte maturation have demonstrated the feasibility of gene overexpression screening in identifying potent regulators of germ cell development. Here we screened 47 folliculogenesis-regulating TFs for their role in hPGCLC and oogonia formation, identifying DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. Additionally, we identify a set of three TFs, ZNF281, LHX8, and SOHLH1 whose combinatorial overexpression drives direct oogonia-like formation from hiPSCs in a four-day, feeder-free monolayer culture condition with additional feeder-free culture capabilities post-isolation. We characterize these TF-based germ cells via gene and protein expression analyses, and demonstrate their broad similarity to in vivo germ cells. Together, these results identify novel regulators of human germ cell development and establish new TF-based tools for human in vitro oogenesis research.

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Maintenance of Human Primordial Germ Cell-Like Cells in a Long-Term Culture System

Gell

Shioda

2023

Methods in Molecular Biology

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Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers

Cited by 13 publications

References 29 publications

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

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

Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors

Maintenance of Human Primordial Germ Cell-Like Cells in a Long-Term Culture System

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