In vitro generation of Sertoli-like and haploid spermatid-like cells from human umbilical cord perivascular cells

Abstract

BackgroundFirst trimester (FTM) and term human umbilical cord-derived perivascular cells (HUCPVCs), which are rich sources of mesenchymal stem cells (MSCs), can give rise to Sertoli cell (SC)-like as well as haploid germ cell (GC)-like cells in vitro using culture conditions that recapitulate the testicular niche.Gamete-like cells have been produced ex vivo using pluripotent stem cells as well as MSCs. However, the production of functional gametes from human stem cells has yet to be achieved.MethodsThree independent lines of FTM and term HUCPVCs were cultured using a novel 5-week step-wise in vitro differentiation protocol recapitulating key physiological signals involved in testicular development. SC- and GC-associated phenotypical properties were assessed by real-time polymerase chain reaction (RT-PCR), quantitative PCR immunocytochemistry, flow cytometry, and fluorescence in-situ hybridization (FISH). Functional spermatogonial stem cell-like properties were assessed using a xenotranplantation assay.ResultsWithin 3 weeks of differentiation, two morphologically distinct cell types emerged including large adherent cells and semi-attached round cells. Both early GC-associated markers (VASA, DAZL, GPR125, GFR1α) and SC-associated markers (FSHR, SOX9, AMH) were upregulated, and 5.7 ± 1.2% of these cells engrafted near the inner basal membrane in a xenograft assay. After 5 weeks in culture, 10–30% of the cells were haploid, had adopted a spermatid-like morphology, and expressed PRM1, Acrosin, and ODF2. Undifferentiated HUCPVCs secreted key factors known to regulate spermatogenesis (LIF, GDNF, BMP4, bFGF) and 10–20% of HUCPVCs co-expressed SSEA4, CD9, CD90, and CD49f. We hypothesize that the paracrine properties and cellular heterogeneity of HUCPVCs may explain their dual capacity to differentiate to both SC- and GC-like cells.ConclusionsHUCPVCs recapitulate elements of the testicular niche including their ability to differentiate into cells with Sertoli-like and haploid spermatid-like properties in vitro. Our study supports the importance of generating a niche-like environment under ex vivo conditions aiming at creating mature GC, and highlights the plasticity of HUCPVCs. This could have future applications for the treatment of some cases of male infertility.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0491-8) contains supplementary material, which is available to authorized users.

Male germline stem cells in non-human primates

“…Recent studies report generation of mesoderm-like cells from human iPS cells, which can be induced to produce primordial germ-like cells . In vitro generation of Sertoli-like cells and haploid spermatids has been reported using human umbilical cord perivascular cells, which demonstrates differentiation potential of these cells (Shlush et al, 2017). Although the functional competence of these in vitro generated cell types still remains to be validated, the transcriptome analysis of in vitro generated human PGCs shows comparable expression patterns to cynomolgus monkey PGCs derived from early amnion .…”

“…For instance, gestation in marmoset monkey takes only 143-145 days, while in macaques it ranges between 160 and 175 days. This is in contrast to human gestation, which takes about 267 days (Silk et al, 1993;Aeckerle et al, 2015). Furthermore, despite this comparatively short gestation period, the processes of PGC specification and migration are still significantly delayed in marmoset monkeys (Phillips, 1976;Merker et al, 1988;Li et al, 2005).…”

Genome editing approaches to augment livestock breeding programs

“…MSCs have been shown to express multiple germ cell markers following treatment with retinoic acid and/ or various growth factors (Dissanayake et al, 2018;Ghasemzadeh-Hasankolaei et al, 2014;Hua et al, 2009;Luo et al, 2019;Shirzeyli et al, 2017;Wei et al, 2016;Yan et al, 2015). Haploid spermatid-like cells have been observed from MSC-derived PGCLC cultures and transplants (Ghasemzadeh-Hasankolaei et al, 2016a,b;Shlush et al, 2017;Zhang et al, 2014). MSCs do not form teratomas when injected into donor testes and have even been shown to help reestablish the testicular niche following chemical damage (Cakici et al, 2013;Maghen et al, 2017;Vahdati et al, 2017).…”

Axon Degeneration Is Rescued with Human Umbilical Cord Perivascular Cells: A Potential Candidate for Neuroprotection After Traumatic Brain Injury

“…HUCPVCs express MSC markers (CD44, CD105, CD146, CD73, CD34) and negative for hematopoietic markers, CD31 and CD45 (macrophage), when propagated in culture to maintain expression of vimentin, alpha smooth muscle actin, and pericyte markers (3G5, NG2, PDGFRb). Investigation of the gene profile of HUCPVCs reveals their potential to secrete neurotrophic [neurotrophin-3 (NT3), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF)] and angiogenic factors [bone morphogeneic protein (BMP1), BMP4, fibroblast growth factor (FGF1), and FGF2] [6][7][8][9]. UC-MSC secretes factors that are conducive to survival and differentiation of immature neurons, indicating their potential to stimulate neurogenesis in vivo [10].…”

“…UC-MSC secretes factors that are conducive to survival and differentiation of immature neurons, indicating their potential to stimulate neurogenesis in vivo [10]. They also contain factors that protect against excitotoxicity and apoptosis to promote survival [9,11,12]. These data taken together point to UC-MSC and HUCPVCs being a promising candidate to study axon breakdown following injury and a potential cell therapy strategy.…”