Zum Potential spermatogonialer Stammzellen
Humangenetik
Zusammenfassung der Projektergebnisse
This project set out to (i) elucidate the role of germ cell (GC)-specific and premeiotic (PrM) genes in transdifferentiation and pluripotency, (ii) improve in vitro spermatogenesis from pluripotent mouse stem cells and transfer the improved strategy to human pluripotent stem cells and (iii) develop methods for the isolation, culture and reprogramming of human spermatogonial stem cells (hSSCs) to pluripotency. We were able to show that stage-specific GC marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency. We further demonstrated that temporal expression of Dppa3 is critical for the generation of fullyreprogrammed induced pluripotent stem cells and for maintaining the Dlk1-Dio3 imprinting during somatic cell reprogramming. The planned improvement of in vitro spermatogenesis from pluripotent mouse stem cells by stable overexpression of the premeiotic gene Stra8 resulted in cells displaying postmeiotic marker gene expression indicative of an earlier entry into and progress through the meiotic process but lacking meiotic or postmeiotic structures in immunostaining and electron microscopy analysis. In addition, imprinted gene methylation analysis of these cells indicated that stable Stra8 overexpression had an effect on genomic imprinting during in vitro spermatogenesis, but did not result in a correct reestablishment of male GC-specific methylation imprints. Using mRNA reprogramming of scrotal human fibroblast (SHF) cell lines, we established three different cryopreserved induced SHF cell lines with no karyotypic abnormalities, whose pluripotent character was proven by alkaline phosphatase and pluripotency marker staining. Attempts to in vitro differentiate these induced SHF cells to haploid GCs could unfortunately not be further followed up due to lack of time. We could further confirm FGFR3 as potential hSSC marker using whole mount preparations of tubuli seminiferi and establish a method combining magnetic cell isolation and micromanipulator-assisted picking of bead-bound single cells for isolation of hSSCs (FGFR3-positive cells). The isolated cells could be maintained in cell culture for up to 70 days and displayed pluripotency marker expression, but could up to now not be expanded under the chosen cell culture conditions. The alternative isolation of hSSCs from testicular biopsies using the Magnetic Activated Cell Sorting technique for the SSC surface marker α6- Integrin succeeded in the enrichment of putative hSSCs exhibiting grape-like structures characteristic of SSCs and a positive staining for the GC marker PLZF, but did not lead to the establishment of a stable and proliferating hSSC line. In the course of our experimental work, we further succeeded in developing an efficient and robust as well as easy and cost-effective system for the generation of early male GCs from mouse embryonic stem cells by co-culture with human testicular cells.
Projektbezogene Publikationen (Auswahl)
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(2008) Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles. Mol Hum Reprod. 14:521-529
Zovoilis A, Nolte J, Drusenheimer N, Zechner U, Hada H, Guan K, Hasenfuss G, Nayernia K, Engel W
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(2009) Comparative methylation profiles and telomerase biology of mouse multipotent adult germline stem cells and embryonic stem cells. Mol Hum Reprod. 15:345-353
Zechner U, Nolte J, Wolf M, Shirneshan K, Hajj NE, Weise D, Kaltwasser B, Zovoilis A, Haaf T, Engel W
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(2009) Multipotent adult germline stem cells and embryonic stem cells: comparative proteomic approach. J Proteome Res. 8:5497-5510
Dihazi H, Dihazi GH, Nolte J, Meyer S, Jahn O, Müller GA, Engel W
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(2010) Embryonic stem cell related miRNAs are involved in differentiation of pluripotent cells originating from the germ line. Mol Hum Reprod 16:793-803
Zovoilis A, Pantazi A, Smorag L, Opitz L, Salinas-Riester G, Wolf M, Zechner U, Holubowska A, Stewart CL, Engel W
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(2010) Global and gene-specific histone modification profiles of mouse multipotent adult germline stem cells. Mol Hum Reprod 17:166-174
Khromov T, Pantakani DV, Nolte J, Wolf M, Dressel W, Engel W, Zechner U
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(2010) Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes. Mol Hum Reprod 16:846-855
Meyer S, Nolte J, Opitz L, Salinas-Riester G, Engel W
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(2010) PSCDGs of mouse multipotent adult germline stem cells can enter and progress through meiosis to form haploid male germ cells in vitro. Differentiation 80:184-94
Nolte J, Michelmann HW, Wolf M, Wulf G, Nayernia K, Meinhardt A, Zechner U, Engel W
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(2010) Screening for biomarkers of spermatogonia within the human testis: a whole genome approach. Hum Reprod 25:1104–1112
von Kopylow K, Kirchhoff C, Jezek D, Schulze W, Feig C, Primig M, Steinkraus V, Spiess AN
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(2011) Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency. PLoS One 6:e22413
Xu X, Pantakani DV, Lührig S, Tan X, Khromov T, Nolte J, Dressel R, Zechner U, Engel W
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(2012) Differential marker expression specifies rarefaction zone-containing human Adark spermatogonia. Reproduction 143:45–57
von Kopylow K, Staege H, Spiess AN, Schulze W, Will H, Primig M, Kirchhoff C
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(2012) Fibroblast growth factor receptor 3 is highly expressed in rarely dividing human type A spermatogonia. Histochemistry and Cell Biology 138:759–772
von Kopylow K, Staege H, Schulze W, Will H, Kirchhoff C
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(2012) Gene Expression and Epigenetic Signatures of Germ Cell-Derived Pluripotent Stem Cells and Embryonic Stem Cells in: Stem Cells and Cancer Stem Cells, Volume 6, Therapeutic Applications in Disease and Injury, Editors: M.A. Hayat, Springer Dordrecht Heidelberg New York London
Nolte J, Pantakani DV, Dihazi H, Zechner U
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(2015) Temporal expression of Dppa3 is critical for generation of fully-reprogrammed iPS cells and for maintaining the Dlk1-Dio3 imprinting during somatic cell reprogramming. Nat Commun 6:6008
Xu X, Smorag L, Nakamura T, Dressel R, Fitzner A, Linke M, Zechner U, Engel W Pantakani DV
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(2016) Isolation and gene expression analysis of single potential human spermatogonial stem cells. Mol Hum Reprod 22:229-39
von Kopylow K, Schulze W, Salzbrunn A, Spiess AN
