Trans differentiating human adipose-derived mesenchymal stem cells into male germ-like cells utilizing Rabbit Sertoli cells: An experimental study

Abstract Background Mesenchymal stem cells (MSCs) are deemed as potential new therapeutic agents for infertility treatment and adipose tissue (AT) becomes a potential MSCs source. To direct MSCs through the differentiation process properly, an environment comparable to the in vivo niche might be indispensable. Objective This study aims to differentiate human AT-derived MScs (hAD-MScs) into male germ-like cells in vitro using a combination of rabbit Sertoli cells conditioned medium (SCCM), bone morphogenetic protein 4, and retinoic acid. Materials and Methods MScs were isolated from human ATs of fertile and infertile donors. The verified MScs were differentiated using a 2-step protocol; the first step included 20 ng/ml bone morphogenetic protein 4 treatment. The second step was performed utilizing 1 μM retinoic acid and/or SCCM. The morphological changes and the expression of germ cell (GC)-specific markers: octamer-binding transcription factor-4; stimulated by retinoic-acid-8, synaptonemal complex protein-3, andprotamine-1 were assessed in the treated cells using quantitative polymerase chain reaction. Results Induction of hAD-MScs resulted in the upregulation of GC-specific genes where SCCM treatment showed the highest expression. The synaptonemal complex protein-3andprotamine-1 gene expression was detected after 19 and 26 days of induction, respectively. PRM1 was detected in hAD-MScs cultured in SCCM earlier than in other treated groups. The treated cells became more elongated-like spindles and formed aggregates. Conclusion hAD-MScs differentiated to GC lineage exhibited the ability to express GC-specific markers under in vitro conditions, and rabbit's Sertoli cells can be used for inducing transdifferentiation of hAD-MScs into germ-like cells.


Introduction
In the past few years, researchers have developed new options to treat infertility. One of the latest options is using stem cells to Some researchers have differentiated human stem cells using mice testicular tissue extract (1,5). It was reported that even though the mouse is the most often used mammalian model for germline research, several features of human PGC development may differ from those of mice. In fact, certain mouse embryonic tissues involved in PGC formation have no obvious human equivalent (6). Therefore, it has shown that the other option is to employ gonadal somatic cells from other animals with flat-disc epiblasts and a molecular network comparable to the human PGC, such as pigs or rabbits (7).
The rabbit is now the second animal, after the mouse, to have a regulatory network involved in germline specification expressed in a realistic time frame throughout early gastrulation and development stages. Rabbit embryology shares numerous similarities with that of humans, most notably in the general appearance of the perigastrulation embryo, which resembles a flat disc rather than the cup-shaped cylinder typical of rodents (8). In addition, it is broadly utilized in medical studies as a paradigm in human male reproductive system toxicology research (9,10). So, in our study, we were concerned with differentiating human MSCs into GCs-like entities in vitro using the conditioned medium prepared from pre-pubertal rabbit testicular tissue that is similar to human tissue for along with exogenous factors that were reported to be the most important and key regulators of the in vivo differentiation processes such as bone morphogenetic protein 4 (BMP4) and retinoic acid (RA).

Isolation and preparation of Sertoli cells (SCs)-conditioned medium
Testes were collected from 42-days-old 5 healthy male prepubertal rabbits of the Dutch Rabbits breed (Oryctolagus Cuniculus) according to the American Veterinary Medical Association guidelines 2020. SCs were isolated using a 2-step enzymatic digestion protocol modified from published protocols (1,11). Testes were then separated and washed 3 times with phosphate buffered saline (PBS) (Biological Industries, USA), they were then minced into small pieces and incubated in 10 ml of 1 mg/mL collagenase type IV (Worthington Biochemical, USA) solution at 37°C for 60 min with shaking (120 cycle/min) then,

Evaluation of hAD-MScs viability after treatment with RA or BMP4
MSCs at the 4 th passage were cultured in 96-wells plate at density of 4500 cells/well in

Gene expression analysis by quantitative RT-PCR
After each period of differentiation treatment into MGCs (19, and 26 days), the cells were trypsinized, and total RNA was isolated from the 4 groups using ISOLATE II Biofluids RNA Phenol Free Kit, its concentration and quality were checked, and 1 μg of total RNA was reverse transcribed to cDNA using SensiFAST TM cDNA Synthesis Kit. Samples were subjected to quantitative polymerase chain reaction using PCR Go Taq® Green Master Mix (Promega, USA) for 40 cycles using the following PCR conditions: 2 min at 95°C followed by 95°C for 5 sec, 59-60°C for 20 sec, and 72°C for 20 sec. Table I

Results
A few hours after enzymatic digestion of rabbit testis and culturing of cell suspension in the culture flask, SCs begin to attach to the bottom of the culture flask; 2 distinct cell phenotypes were detected after 24 hr, round GCs and fibroblastlike somatic SCs adhered to the bottom of the culture flask ( Figure 1A and 1B). On day 2, SCs began to flatten, spread out, and had a fibroblastic appearance. They proliferated immensely within 4-7 days and formed a monolayer. The GCs were removed by exchanging of culture medium every day. After passage, the cells made monolayer entity at the bottom of culture flask and showed irregular shapes with predominately fibroblastic morphology ( Figure 1C).
The morphological characteristics of cultured SCs were examined under inverted and light microscope after staining with Leishman stain. In general, the SCs had irregular polygonal shapes, well-formed cytoplasmic extensions with a granular appearance in the cytoplasm, large nuclei, and 3 nucleoli, they made elongations, flattened, and attempted to create contact with the other cells ( Figure 1D).
MSCs were isolated from human AT. After one day of culturing, MSCs exhibited the capacity International Journal of Reproductive BioMedicine AD-MScs to adhere to the plastic culture flask's bottom, they were initially round and begin to adhere to the bottom of the culture flask ( Figure 2A) and some spindle-shaped cells appeared among the mononuclear cells on day 2. On day 4, cells began to flatten, spread out, and take a fibroblastic appearance, and the morphology did not change during cell passages ( Figure 2B). They started to proliferate within 5-7 days and formed a monolayer with a uniform fibroblast-like morphology ( Figure  2C) after staining with Leishman stain. The SCs appeared to have irregular polygonal shapes, wellformed cytoplasmic extensions with a granular appearance in the cytoplasm, large nuclei, and 3 nucleoli, they produced extensions, they were flattened and attempted to make contact with other cells ( Figure 2D).

hAD-MScs surface markers evaluation
The results indicated that hAD-MScs expressed the MSCs markers CD73, CD105, and CD90, but CD45, the hematopoietic stem cell marker, was not expressed (Figure 3). All the surface marker genes expression assessed in the isolated AD-MSCs conformed to the criteria of MSCs established earlier.

Effect of various doses of RA on the hAD-MScs viability
The MTT test findings revealed that at 10 −4 M RA, compared to doses of 10 −5 , 10 −6 , and 10 −7 M RA, the percentage of cell viability was considerably lower

Effect of various doses of BMP4 on the hAD-MScs viability
The MTT test findings revealed at dosage 5 ng/ml BMP4 compared with dosages of 10, 25, 50, and 100 ng/ml BMP4 and control groups, the percentage of cell viability was considerably lower (p < 0.01). In addition, a significant increase was observed in viability in 50 and 100 ng/ml compared to 10 and 25 ng/ml BMP4 and control groups (p < 0.01). However, no considerable difference was observed in the cell viability percentage between doses of 10, 25 ng/ml BMP4 and the control group (p = 0.36). A value between 10 and 25 ng/ml BMP4 dosages, 20 ng/ml was selected for induction of differentiation of MSCs into germlike cells ( Figure 5).

Cell morphology evaluation in the treatment groups
The change in cell morphology was evaluated every 3 days using an inverted phase contrast microscope. Figure 6 represents morphological characteristics of the hAD-MScs before treatment with 20 ng/ml BMP4 (Figures 6A, B) and after the cells were cultured in a dosage of 20 ng/ml BMP4 ( Figures 6C, D), these cells were compared with the control group morphologically after 5 days of culture. The morphological change was noticeable after culturing in BMP4, such as some cells were more oval unlike fibroblastic cells and formed cell aggregates ( Figures 6C, D). the proliferation of untreated hAD-MScs on days 7, 10, 19, and 26 respectively. While in the treated groups, the cell proliferation in the RA treated group was lower than the other treatment groups and untreated group after 7 days. In addition, the morphology of hAD-MScs gradually altered from fibroblast-like to be more oval with clear cellular boundaries and have a tadpole-like shape after 7 days ( Figures 7E and 8E), which in the following induction times did not alter considerably ( Figures  7 F-H and 8 (F-H)).
hAD-MScs treated with RA/SCCM showed higher proliferation, and most of the cells retained their fibroblastic appearance at day 7 ( Figures   7I and 8I). Their fibroblastic shape changed to become like slender spindles, and a few cells that resembled tadpoles were present within 10-12 days ( Figures 7J and 8J), which transformed into slender spindles in subsequent times (Figures 7  and 8 (K-L)).
In the SCCM treated group, virtually most of the cells lengthened and took the shape of spindles within 7 days, the cells appeared to be more elongated than in RA/SCCM group, and the presence of aggregates increased in subsequent culture times (Figures 7 and 8 (M-P)).

Expression of GC specific genes in treated hAD-MScs
The expression levels of GCs specific markers, Interestingly, treatment of hAD-MScs with RA, RA/SCCM, and SCCM led to a decrease in OCT4 expression at day 19 compared to the control group ( Figure 10A), a significant decrease was shown in RA/SCCM and SCCM groups (p = 0.01). While at day 26, the OCT4 expression was significantly higher in the RA/SCCM and SCCM groups compared to control (p = 0.01) ( Figure 10B). However, no significant change was observed in OCT4 expression between 19 and 26 days of treatment (p = 0.30).
The SCP3 was shown to be a mitotic marker whose expression is essential for the development of the synaptonemal complex in homologous chromosomes and was detected following treatment of hAD-MScs with RA, RA/SCCM, and SCCM after 19 days of treatment when compared to the untreated hAD-MScs. On day 19, the group receiving RA treatment had significantly higher levels of SCP3 expression than the other groups (p = 0.03). However, the difference was not statistically significant in RA/SCCM and SCCM groups (p = 0.93) (Figures 10C and D).
The expression of SCP3 was found to be significantly higher on day 26 in the SCCM group compared to other groups in fertile donor treated cells (p = 0.02) ( Figure 10C), while in infertile donor treated cells, the SCP3 expression was higher in RA treated group ( Figure 10D). Although the overall expression of SCP3 was not significantly different between 19 and 26 days of treatment (p = 0.14).
Similarly, the post-meiotic spermatid marker however, compared to other groups, the SCCMtreated group had a high expression level (p = 0.04) ( Figure 10F). Total expression differences of PRM1 between 19-and 26-days treatment were not statistically significant (p = 0.06). The expression of the pre-meiotic marker, STRA8 was not detected neither in treated nor in untreated groups after 19 and 26 days of treatment (Figures 9A and  B).     The viability of the cells was determined using MTT assay and the concentration of BMP4 that had the least effects on the cell's viability was selected. The percentage of cell viability was significantly decreased in dosage of 5 ng/ml BMP4 compared with dosages of 10, 25, 50, and 100 ng/ml BMP4 and control groups. In addition, there is a significant increase in viability in 50 and 100 ng/ml compared to 10 and 25 ng/ml BMP4 and control groups. However, percentage of cell viability was not significantly different between dosages of 10, 25 ng/ml BMP4 and the control group.  . The morphology of hAD-MScs gradually altered from fibroblast-like to be more oval with clear cellular boundaries and have a tadpole-like shape after 7 days, which in the following induction times did not alter considerably. I-L) hAD-MScs were treated with bone morphogenetic protein 4 (BMP4) then retinoic acid (RA) in combination with Sertoli cell conditioned medium (SCCM). Cells fibroblastic shape changed to become like slender spindles and a few cells that resembled tadpoles were present within 10-12 days, which transformed to slender spindles in subsequent times. M-P) hAD-MScs were treated with only Sertoli cell conditioned medium (SCCM). Virtually most of the cells lengthened and took the shape of spindles within 7 days, the cells appeared to be more elongated than in RA/SCCM group. (Mag: ×200), scale bar: 1000 µm.

International Journal of Reproductive BioMedicine
Khudair et al. . The morphology of hAD-MScs gradually altered from fibroblast-like to be more oval with clear cellular boundaries and have a tadpole-like shape after 7 days, which in the following induction times did not alter considerably. I-L) hAD-MScs were treated with bone morphogenetic protein 4 (BMP4) then retinoic acid (RA) in combination with Sertoli cell conditioned medium (SCCM). Cells fibroblastic shape changed to become like slender spindles and a few cells that resembled tadpoles were present within 10-12 days, which transformed to slender spindles in subsequent times. M-P) hAD-MScs were treated with only Sertoli cell conditioned medium (SCCM). Virtually most of the cells lengthened and took the shape of spindles within 7 days, the cells appeared to be more elongated than in RA/SCCM group. (Mag: ×200), scale bar: 1000 µm.

Discussion
Stem cells are deemed as prospective innovative therapeutic mechanisms for treating infertility because of their limitless sources and high differentiation capacity (13). Therefore, in this OCT4 is a member of the family of POU transcription factors, is critical for controlling pluripotency throughout embryogenesis and present in both early GCs and pluripotent stem cells (19). OCT4 has been identified as a pluripotency marker, and its expression might be utilized to assess GC differentiation (16). The SCP3 is a late meiosis-specific molecular construction that serves as a molecular framework for chromosomal synapsis regulation (20). STRA8, an RA target gene, has been identified as a GC marker that is expressed mostly during International Journal of Reproductive BioMedicine AD-MScs the pre-meiotic stage of developing MGCs, its mRNA levels are reported to be low or absent in post-meiotic cells (25). Also it has been reported that STRA8 was only observed during the transition of spermatogonia to spermatocytes (26). Ghorbanlou, with co-workers have showed that STRA8 expression was detected after 7 days of treatment of BM-MScs with RA and testicular cells (27). In addition, Dissanayake and co-authors have mentioned that slight STRA8 expression was observed after 2 wk of induction (23), while in our study we evaluate the expression after approximately 3-4 wk, and we did not detect any expression of STRA8.
In the current work, we did not recognize any

Conclusion
In conclusion, this work shows that hAD-MScs