Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys

Lihua Dong; Stine Gry Kristensen; Simone Hildorf; Murat Gul; Erik Clasen-Linde; Jens Fedder; Eva R Hoffmann; Dina Cortes; Jorgen Thorup; Claus Yding Andersen

doi:10.3389/fphys.2019.01155

Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys

Lihua Dong, Stine Gry Kristensen, Simone Hildorf, Murat Gul, Erik Clasen-Linde, Jens Fedder, Eva R Hoffmann, Dina Cortes, Jorgen Thorup, Claus Yding Andersen

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Abstract

Background: Gonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity.

Materials and Methods: A minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.

Results: Proliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.

Conclusion: This study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.

Original language	English
Article number	01155
Journal	Frontiers in Physiology
Volume	10
Pages (from-to)	1-11
Number of pages	11
ISSN	1664-042X
DOIs	https://doi.org/10.3389/fphys.2019.01155
Publication status	Published - 19 Sept 2019

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@article{7910b5fc23244ba8a7f7fd7969b5eb0e,

title = "Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys",

abstract = "Background: Gonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity.Materials and Methods: A minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.Results: Proliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.Conclusion: This study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.",

author = "Lihua Dong and Kristensen, {Stine Gry} and Simone Hildorf and Murat Gul and Erik Clasen-Linde and Jens Fedder and Hoffmann, {Eva R} and Dina Cortes and Jorgen Thorup and Andersen, {Claus Yding}",

note = "Copyright {\textcopyright} 2019 Dong, Kristensen, Hildorf, Gul, Clasen-Linde, Fedder, Hoffmann, Cortes, Thorup and Andersen.",

year = "2019",

month = sep,

day = "19",

doi = "10.3389/fphys.2019.01155",

language = "English",

volume = "10",

pages = "1--11",

journal = "Frontiers in Physiology",

issn = "1664-042X",

publisher = "Frontiers Media S.A.",

}

TY - JOUR

T1 - Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys

AU - Dong, Lihua

AU - Kristensen, Stine Gry

AU - Hildorf, Simone

AU - Gul, Murat

AU - Clasen-Linde, Erik

AU - Fedder, Jens

AU - Hoffmann, Eva R

AU - Cortes, Dina

AU - Thorup, Jorgen

AU - Andersen, Claus Yding

PY - 2019/9/19

Y1 - 2019/9/19

N2 - Background: Gonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity.Materials and Methods: A minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.Results: Proliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.Conclusion: This study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.

AB - Background: Gonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity.Materials and Methods: A minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.Results: Proliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.Conclusion: This study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.

U2 - 10.3389/fphys.2019.01155

DO - 10.3389/fphys.2019.01155

M3 - Journal article

C2 - 31607938

SN - 1664-042X

VL - 10

SP - 1

EP - 11

JO - Frontiers in Physiology

JF - Frontiers in Physiology

M1 - 01155

ER -

Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys

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