Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

Hindawi Publishing Corporation Stem Cells International Volume 2016, Article ID 8582526, 17 pages http://dx.doi.org/10.1155/2016/8582526 Research Article Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells Sabine Conrad,1 Hossein Azizi,2,3,4 Maryam Hatami,2 Mikael Kubista,5,6 Michael Bonin,7 Jörg Hennenlotter,8 Karl-Dietrich Sievert,8 and Thomas Skutella2 1 Sabine Conrad, P.O. Box 12 43, 72072 Tübingen, Germany Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany 3 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395, Tehran 4644, Iran 4 Faculty of Biotechnology, Amol University of Special Modern Technologies, P.O. Box 46168, Amol 49767, Iran 5 TATAA Biocenter AB, Odinsgatan 28, 41103 Göteborg, Sweden 6 Institute of Biotechnology at the Czech Academy of Sciences Videnska 1083, 14220 Prague 4, Czech Republic 7 Institute of Anthropology and Human Genetics, Microarray Facility, University Clinic, Calwerstraße 7, 72076 Tübingen, Germany 8 Department of Urology, University of Tübingen Hospital, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany 2 Correspondence should be addressed to Thomas Skutella; Received 3 December 2014; Revised 9 February 2015; Accepted 11 February 2015 Academic Editor: Irma Virant-Klun Copyright © 2016 Sabine Conrad et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers. 1. Background Human adult germ stem cells (haGSCs) derived from highly enriched spermatogonia isolated from adult human testicular tissue were shown to be highly versatile and having some similarities with human embryonic stem cells (hESCs), including the expression of genes associated with pluripotent cells and the ability to be in vitro differentiated into a number of cell lineages comprising the three germ layers [1–6]. In the studies of Mizrak et al. [5], Chikhovskaya et al. [7], and Gonzalez et al. [8], the cells expressing markers of pluripotency were probably derived from mesenchymal stem cells (MSCs) or were more MSC-like. Moreover, it has also been proposed that haGSCs may be low-differentiated testicular fibroblasts [9]. In contrast, Stimpfel et al. [10] demonstrated that both germ- and mesenchyme-derived stem cells were present in stem cell clusters from human testis biopsy, which could differentiate into cells of all three germ 2 layers. Recently, Lim et al. [6] provided evidence that haGSCs show similarities to hESCs and are being able to generate small teratomas. The findings of all these studies raised some new questions about the real character of pluripotency in haGSCs. It is generally accepted that pluripotency of cells requires the activation of a transcriptional regulatory network [11], a phenomenon which has been observed in ex vivo cultures of early embryonic cells and also in cells of the germ cell lineage, in which members of the pluripotency network are normally active, including embryonic cells during development of morula and blastocyst-stage (inner cell mass) embryo, epiblast, primordial germ cells (PGCs), and germline stem cells. One main step in analyzing the biology of haGSCs and pluripotency in adult stem cells is to determine their germ cell-specific gene expression profile. The present knowledge regarding the molecular markers that define haGSCs and their pluripotency is significantly limited. Therefore, the goal of this study was to investigate the molecular profile of haGSCs, which are able to comprise both the expression of a residual germ cell profile and genes related to pluripotency, in addition to our previous study on hSSCs [1]. In order to accomplish this goal, we sought to compare the gene expression profiles of haGSCs generated from shortterm cultured enriched spermatogonial stem cells (hSSCs) to hFibs and hESCs using (1) single cell nanofluid realtime PCR (Fluidigm) of a representative haGSC colony, (2) microarray analysis, and (3) Fluidigm real-time PCR and immunohistochemistry of haGSC colonies to validate the microarray data. Here, we show that haGSCs are adult stem cells with a specific molecular profile, which is related to spermatogonia. Under hESC culture conditions they can be selected and cultured and maintain a state resembling in part gene expression related to the expression patterns found in pluripotent cells. 2. Results 2.1. Generation of haGSC Colonies from Enriched Fraction of Spermatogonia. Colonies or clusters of haGSC developed spontaneously from the CD49f MACS and matrix (collagen nonbinding, laminin binding) selected fraction of enriched spermatogonia (Figure 1) but not from the negative selected fraction of cells or from patients without spermatogonia. By MACS and matrix selection, the hFibs, which overgrow the primary cell cultures, were depleted and remained in the nonselected populations of cells. The hFibs appeared morphologically completely different compared to haGSCs (Figure 1). In the primary cultures, the first small haGSC colonies/islands started to appear 4–6 weeks after culture of enriched spermatogonia in hGSC medium. The denser haGSC aggregations were manually selected for further propagation and characterization (Figure 1(d)). The typical haGSC colony consisted of central part of colony and outgrowing epithelial cells resembling early cell colonies of hESCs (Figure 1(e)). In the negatively selected cell fraction, no epithelial haGSC colony formation was observed [9]. This Stem Cell (...truncated)