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Potential stemness of frozen-thawed testicular biopsies without sperm in infertile men included into the in vitro fertilization programme.

Stimpfel M, Skutella T, Kubista M, Malicev E, Conrad S, Virant-Klun I - J. Biomed. Biotechnol. (2012)

Bottom Line: The patients were diagnosed with Sertoli Cell-Only Syndrome alone or combined with maturation arrest.High throughput real-time quantitative PCR followed by multivariate analysis revealed the formation of distinct cell clusters reflecting high degree of similarity and some of these cell clusters expressed the genes characteristic for pluripotent stem cells.In the presence of the follicular fluid, prepared as serum, putative testicular stem cells showed a certain degree of plasticity, and spontaneously differentiated into adipose-like and neuronal-like cells.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Unit, Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, 1000 Ljubljana, Slovenia.

ABSTRACT
We describe the potential stemness of a small amount of frozen-thawed testicular tissue without sperm obtained by biopsy from six patients undergoing assisted reproductive treatment. The patients were diagnosed with Sertoli Cell-Only Syndrome alone or combined with maturation arrest. Trying to provide the natural stem cell niche for cultured stem cells, all isolated cells from enzymatically degraded biopsies where cultured together in different culture media and the presence of putative mesenchymal and putative pluripotent ES-like stem cells was indicated using different methods. High throughput real-time quantitative PCR followed by multivariate analysis revealed the formation of distinct cell clusters reflecting high degree of similarity and some of these cell clusters expressed the genes characteristic for pluripotent stem cells. In the presence of the follicular fluid, prepared as serum, putative testicular stem cells showed a certain degree of plasticity, and spontaneously differentiated into adipose-like and neuronal-like cells. Additionally, using differentiation protocols putative testicular stem cells were differentiated into neuronal- and pancreatic-like cells. This study shows that in assisted reproduction programmes, testicular tissue with no sperm might be an important source of stem cells, although it is discarded in daily medical practice; this requires further research.

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Testicular cell clusters and their gene expression analyses. (a) TSC1. (b) TSC2. (c) TSC3. (d, e) TSC4. (inverted microscope, Hoffman, with/without Autowhite). (f) Heatmap comparing genes' expressions in testicular cell clusters with human embryonic stem cells and fibroblasts. Scale Bar: (a–e) is 100 μm.
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fig10: Testicular cell clusters and their gene expression analyses. (a) TSC1. (b) TSC2. (c) TSC3. (d, e) TSC4. (inverted microscope, Hoffman, with/without Autowhite). (f) Heatmap comparing genes' expressions in testicular cell clusters with human embryonic stem cells and fibroblasts. Scale Bar: (a–e) is 100 μm.

Mentions: Four samples of 6 testicular cell clusters were isolated from one cell culture: TSC1—one cluster cultured for 14 days in DMEM/F-12 medium with follicular fluid, TSC2—one cluster cultured for 14 days in hESC medium with follicular fluid, TSC3—two clusters cultured for 140 days in DMEM/F-12 medium with follicular fluid, and TSC4—two clusters cultured for 140 days in DMEM/F-12 medium with follicular fluid. Gene expression in these samples was compared with control groups based on human embryonic stem cells—H1 line (150 and 200 cells) and human fibroblasts (150 and 200 cells). Cluster TSC1 (Figure 9(a)) strongly expressed a variety of genes related to pluripotency. In culture, small round cells with yellow colour and a diameter of up to 5 μm appeared close to this cluster (Figure 9(b)). Also the other cell clusters (Figures 10(a)–10(e)) expressed some of the genes characteristic of pluripotency, but to lower extent than TSC1 (Figure 10(f)). Gene expression in the putative testicular stem cells (TSCs) was comparable to that in the human embryonic stem cells (hESC), but was quite different from the expression in the human fibroblasts, as reflected by the heatmap (Figure 10(f)), corresponding dendrogram (Figure 11(a)), PCA clustering (Figure 11(b)), and univariate analysis of the genes summarized by descriptive statistics (Figure 12); TSCs clustered with the hESCs and in the PCA they have similar PC1 score; they differ, however, in the PC2 (Figure 11(b)). The fibroblasts were clearly different. Comparing genes' expression between putative TSCs and hESCs and using Bonferroni correction for multiple testing, we found significantly lower expression of DNMT3B in TSC's (P = 0.00026). Genes DAZL, NANOS, KLF4, DNMT1, STELLA, NANOG, STAT3, OCT4A, and GPR125 were differentially expressed at P < 0.05 but cannot be considered significant without validation because of the large number of genes compared (Figure 12(a)). On the other hand, fibroblasts showed quite different expression; in particular they did not express many of the genes related to pluripotency. Comparing with TSCs they underexpress OCT4A (P = 0.00187), NANOG (P = 0.00206), SOX-2 (P = 0.00145), and NANOS (P = 0.00247) (Figure 12(b)). Comparing with hESCs they underexpress OCT4A (P = 0.00108), OCT4B (P = 0.00045), NANOG (P = 0.00151), SOX-2 (P = 0.00014), DNMT3B (P = 0.00106), and CDH1 (P = 0.00066). One-way ANOVA confirmed that the variation in genes' expression among the groups TSCs, hESCs, and fibroblasts was larger than expected by chance for OCT4A (P = 0.00040), LIN28 (P = 0.00170), GDF3 (P = 0.00012), NANOG (0.00044), SOX-2 (P = 0.00044), TDGF1 (P = 0.00154), DNMT3B (P = 0.00011), TERT (P = 6.068E − 5), NANOS (P = 0.00126), and CDH1 (P = 2E − 8). These results were consistent with the observations by flow cytometry and immunocytochemistry.


Potential stemness of frozen-thawed testicular biopsies without sperm in infertile men included into the in vitro fertilization programme.

Stimpfel M, Skutella T, Kubista M, Malicev E, Conrad S, Virant-Klun I - J. Biomed. Biotechnol. (2012)

Testicular cell clusters and their gene expression analyses. (a) TSC1. (b) TSC2. (c) TSC3. (d, e) TSC4. (inverted microscope, Hoffman, with/without Autowhite). (f) Heatmap comparing genes' expressions in testicular cell clusters with human embryonic stem cells and fibroblasts. Scale Bar: (a–e) is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303891&req=5

fig10: Testicular cell clusters and their gene expression analyses. (a) TSC1. (b) TSC2. (c) TSC3. (d, e) TSC4. (inverted microscope, Hoffman, with/without Autowhite). (f) Heatmap comparing genes' expressions in testicular cell clusters with human embryonic stem cells and fibroblasts. Scale Bar: (a–e) is 100 μm.
Mentions: Four samples of 6 testicular cell clusters were isolated from one cell culture: TSC1—one cluster cultured for 14 days in DMEM/F-12 medium with follicular fluid, TSC2—one cluster cultured for 14 days in hESC medium with follicular fluid, TSC3—two clusters cultured for 140 days in DMEM/F-12 medium with follicular fluid, and TSC4—two clusters cultured for 140 days in DMEM/F-12 medium with follicular fluid. Gene expression in these samples was compared with control groups based on human embryonic stem cells—H1 line (150 and 200 cells) and human fibroblasts (150 and 200 cells). Cluster TSC1 (Figure 9(a)) strongly expressed a variety of genes related to pluripotency. In culture, small round cells with yellow colour and a diameter of up to 5 μm appeared close to this cluster (Figure 9(b)). Also the other cell clusters (Figures 10(a)–10(e)) expressed some of the genes characteristic of pluripotency, but to lower extent than TSC1 (Figure 10(f)). Gene expression in the putative testicular stem cells (TSCs) was comparable to that in the human embryonic stem cells (hESC), but was quite different from the expression in the human fibroblasts, as reflected by the heatmap (Figure 10(f)), corresponding dendrogram (Figure 11(a)), PCA clustering (Figure 11(b)), and univariate analysis of the genes summarized by descriptive statistics (Figure 12); TSCs clustered with the hESCs and in the PCA they have similar PC1 score; they differ, however, in the PC2 (Figure 11(b)). The fibroblasts were clearly different. Comparing genes' expression between putative TSCs and hESCs and using Bonferroni correction for multiple testing, we found significantly lower expression of DNMT3B in TSC's (P = 0.00026). Genes DAZL, NANOS, KLF4, DNMT1, STELLA, NANOG, STAT3, OCT4A, and GPR125 were differentially expressed at P < 0.05 but cannot be considered significant without validation because of the large number of genes compared (Figure 12(a)). On the other hand, fibroblasts showed quite different expression; in particular they did not express many of the genes related to pluripotency. Comparing with TSCs they underexpress OCT4A (P = 0.00187), NANOG (P = 0.00206), SOX-2 (P = 0.00145), and NANOS (P = 0.00247) (Figure 12(b)). Comparing with hESCs they underexpress OCT4A (P = 0.00108), OCT4B (P = 0.00045), NANOG (P = 0.00151), SOX-2 (P = 0.00014), DNMT3B (P = 0.00106), and CDH1 (P = 0.00066). One-way ANOVA confirmed that the variation in genes' expression among the groups TSCs, hESCs, and fibroblasts was larger than expected by chance for OCT4A (P = 0.00040), LIN28 (P = 0.00170), GDF3 (P = 0.00012), NANOG (0.00044), SOX-2 (P = 0.00044), TDGF1 (P = 0.00154), DNMT3B (P = 0.00011), TERT (P = 6.068E − 5), NANOS (P = 0.00126), and CDH1 (P = 2E − 8). These results were consistent with the observations by flow cytometry and immunocytochemistry.

Bottom Line: The patients were diagnosed with Sertoli Cell-Only Syndrome alone or combined with maturation arrest.High throughput real-time quantitative PCR followed by multivariate analysis revealed the formation of distinct cell clusters reflecting high degree of similarity and some of these cell clusters expressed the genes characteristic for pluripotent stem cells.In the presence of the follicular fluid, prepared as serum, putative testicular stem cells showed a certain degree of plasticity, and spontaneously differentiated into adipose-like and neuronal-like cells.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Unit, Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, 1000 Ljubljana, Slovenia.

ABSTRACT
We describe the potential stemness of a small amount of frozen-thawed testicular tissue without sperm obtained by biopsy from six patients undergoing assisted reproductive treatment. The patients were diagnosed with Sertoli Cell-Only Syndrome alone or combined with maturation arrest. Trying to provide the natural stem cell niche for cultured stem cells, all isolated cells from enzymatically degraded biopsies where cultured together in different culture media and the presence of putative mesenchymal and putative pluripotent ES-like stem cells was indicated using different methods. High throughput real-time quantitative PCR followed by multivariate analysis revealed the formation of distinct cell clusters reflecting high degree of similarity and some of these cell clusters expressed the genes characteristic for pluripotent stem cells. In the presence of the follicular fluid, prepared as serum, putative testicular stem cells showed a certain degree of plasticity, and spontaneously differentiated into adipose-like and neuronal-like cells. Additionally, using differentiation protocols putative testicular stem cells were differentiated into neuronal- and pancreatic-like cells. This study shows that in assisted reproduction programmes, testicular tissue with no sperm might be an important source of stem cells, although it is discarded in daily medical practice; this requires further research.

Show MeSH
Related in: MedlinePlus