Expression of genes related to germ cell lineage and pluripotency in single cells and colonies of human adult germ stem cells
Sabine Conrad1, Hossein Azizi3, Maryam Hatami2, Mikael Kubista4, Michael Bonin5, Jörg Hennenlotter6, K.-D. Sievert6 and Thomas Skutella2
1 P.O. Box 1243, 72072 Tübingen, Germany
2Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany
3Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran
4TATAA Biocenter AB, Odinsgatan 28, 41103 Göteborg, Sweden and Institute of Biotechnology at the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic
5Institute of Anthropology and Human Genetics, Microarray Facility, University Clinic, Calwerstraße 7, 72076 Tübingen, Germany
6Department of Urology, University of Tübingen Hospital, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany
This research intended to explain the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which are naturally growing from the CD49f MACS- and matrix (collagen–/laminin+ binding) selected fraction of enriched human spermatogonia.
Features similar to human embryonic stem cells (hESCs) and with a major difference to somatic human fibroblasts (hFibs) of a characteristic germ- and pluripotency-associated gene expression profile of haGSCs were disclosed with the Fluidigm BioMark system single-cell transcriptional profiling of a long-term cultured haGSC cluster, in comparison to hESCs and hFibs. Furthermore, the disparity of haGSC colonies showing gene expression heterogeneity with more or less pluripotency has been proven by genome-wide comparisons with microarray analysis. As study results validate, haGSCs are adult stem cells with a specific molecular gene expression profile in vitro. They resemble, but are not identical with authentic pluripotent stem cells. The haGSC colonies are selectable under ESC conditions and sustain a partly pluripotent state at the molecular level. This level can resemble their cell plasticity and their differentiation capability to split into cells of all germ layers. As a heterogeneous population of cells, the haGSCs differentiate from hFibs or MSCs and express a lower or higher degree of pluripotency in comparison to hESCs.
The catalogue of significantly regulated genes in haGSCs in relation to hFibs contained several highly expressed genes in hESCs, as for instance CD24, EPCAM, L1TD1, SALL4, JARID2, DNMT3A, HOOK1, ACTIVIN A receptor 1B, and REX1. It was validated by Fluidigm RT-PCR, and for CD24 by immunohistochemistry, that the upregulation of these genes in haGSCs is relevant.
For the improval of culture conditions, additional studies are necessary to stop the molecular block hindering the haGSCs from fully converting to molecular pluripotent stem cells.
The importance of this study is several-fold.
haGSCs arise from the fortified population of CD49f MACS and matrix-selected spermatogonia during cell culture (2). They never have their origin in the negatively selected amount or from patients lacking spermatogonia, e.g. Sertoli-Only-Syndrom.
Detecting the difference in haGSC colonies and hFibs was easy, as they are much alike the early hESC colonies. These early hESC colonies exemplify by a pivotal cluster with overgrowing epithelial cells. The earliest diminutive haGSC colonies in the primary cultures began to manifest 4-6 weeks after initiating a culture of enriched spermatogonia in haGSC medium. This was followed by a manual selection of denser haGSC aggregations for continued propagation and definition. It was exemplary for haGSC colonies to comprise the central part of the colony and exuberant epithelial cells akin to incipient cell colonies of hESCs.
The considerable difference of haGSCs from hFibs was exhibited in single-cell Fluidigm analysis concerning the expressing of germ- and pluripotency-associated genes. Some common features of hFibs were found just with a few outliner hESCs and haGSCs, yet the majority of them had nothing in common.
By contrasting of single hESCs with haGSCs, hESCs increasingly expressed a majority of the pluripotency-related genes such as SOX2, NANOG, LIN28, LIN28B, GDF3, CADH1, OCT4a, TDGF1 and UTF1. Besides, haGSCs expressed the germ cell-related genes CD9, GFRA1, NANOS, STAT3, TSPYL, GPR125 and MYC more intensely. A resemblant profile with lower expression levels was revealed when evaluating all haGSCs. This signifies, that the basic pluripotency-related genes including OCT4a, NANOG, SOX2 and LIN28 were expressed by haGSCs. The haGSCs still kept a germ cell-related gene expression profile to some extent. Comparing haGSCs to hESCs, their expression of the core pluripotency-related genes was less intense. However, it proved to be substantially higher than in hFibs.
The heterogeneity of haGSC colonies was revealed, as they shared characteristics with an ESC-like state of pluripotency to a greater or lesser extent. Additionally, different haGSC colonies proved to be relatively heterogeneous regarding their expressing of germ- und pluripotency-associated genes in the microarray study. An aggregate analysis of transcriptome and high variance genes in haGSCs in comparison with hESCs and hFibs displayed the separation of haGSCs from hFibs and their depicting of a particular cell population.
The pathway leading to pluripotency initiated from germ cells by means of natural programming without involving additional factors is also supposedly related to age. An animal study proved that a derivation of ESC-like cells from mouse SSC cultures is practicable before reaching adolescence or up to 7 weeks of age of the mouse (3). However, we referred to the impossibility to derive these cells from mature mice. True adult mouse SSCs seem to be unable to develop into a pluripotent state. With advancing age, this inability is paralleled by a decline in expression of the core pluripotency genes Oct4, Nanog and Sox2 in SSCs. A proliferation of the genes of the spermatogonial differentiation pathway is found simultaneously. If we transferred the information obtained in mice to human beings, the testis tissue would have to be retrieved from boys up to 11-12 years of age reaching the initial phase of puberty.
Of great interest could be the application of boosting factors, such as small molecules, possibly enhancing the probability of SSC to PSC conversion and enlarging the restricted time window of the process.
1: Conrad S, Azizi H, Hatami M, Kubista M, Bonin M, Hennenlotter J, Sievert KD, Skutella T. Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells. Stem Cells Int. 2016;2016:8582526. doi: 10.1155/2016/8582526. Epub 2015 Nov 8. PubMed PMID: 26649052; PubMed Central PMCID: PMC4655073.
2: Conrad S, Azizi H, Hatami M, Kubista M, Bonin M, Hennenlotter J, Renninger M, Skutella T. Differential gene expression profiling of enriched human spermatogonia after short- and long-term culture. Biomed Res Int. 2014;2014:138350. doi: 10.1155/2014/138350. Epub 2014 Mar 12. PubMed PMID: 24738045; PubMed Central PMCID: PMC3971551.
3: Azizi H, Conrad S, Hinz U, Asgari B, Nanus D, Peterziel H, Hajizadeh Moghaddam A, Baharvand H, Skutella T. Derivation of Pluripotent Cells from Mouse SSCs Seems to Be Age Dependent. Stem Cells Int. 2016;2016:8216312. doi: 10.1155/2016/8216312. Epub 2015 Nov 9. PubMed PMID: 26664410; PubMed Central PMCID: PMC4655302.
Acknowledgements: This study was supported by BMBF- project 01GN0821