The sole analysis of testicular cell suspensions by FCM has contributed in many relevant aspects to research and clinics, as exemplified in the previous sections. Whenever the available FCM equipment is also a sorter, any well-defined population in the dot plots can be chosen and classified by fluorescence activated cell sorting (FACS) for downstream studies. The strategies for the distinction and subsequent sorting of spermatogenic cell populations have been varied, ranging from multiparametric analyses that rely on differences in DNA content and light scattering, to the employment of specific antibodies against the stages of interest. The contributions of FCM sorting to the molecular understanding of spermatogenesis have been diverse and important and will be outlined in the following sections.
5.1. Purification of Spermatogenic Cell Populations for Transcriptomic Studies
A deeper understanding of the transcriptional program associated to the spermatogenic process has been a long-dated aim, since it has many potential applications such as diagnosis of infertility, in vitro gamete production for research and infertility treatment [
82], and contraception developments [
83]. Different strategies have been used to overcome the complexity of the male gonad and address transcriptomic studies along spermatogenesis, from the use of whole testes of juvenile animals at increasing ages and analysis of bulk RNA [
23,
25,
28,
83,
84,
85], to the isolation of testicular cell populations by STA-PUT or centrifugal elutriation either for microarray [
25,
86,
87,
88,
89] or RNAseq profiling [
90,
91,
92,
93].
As mentioned above, FCM allows the discrimination of a higher number of spermatogenic cell types and enables the obtainment of highly pure cell populations via FACS [
20,
45]; therefore, it presents important advantages for transcriptomic studies. Based on the FCM profiles of testicular cell suspensions stained with the vital dye Ho342 [
45,
51], Fallahi and colleagues (2010) were able to isolate by FACS an important number of spermatogenic germ cell populations from adult male mice with > 95% purity in all cases. They employed the sorted spermatogenic cell fractions in microarray chips for massive transcriptome profiling, representing a pioneer study in the combination of FCM with transcriptomic studies [
37] (
Figure 8). More recently, we applied our VDG-based sorting protocol [
49,
50] for RNA seq of four testicular cell populations, including two meiotic prophase I populations (L/Z and P/D) (see
Figure 2B). The high purity of the sorted fractions (>95%), combined with RNAseq technology, enabled accurately establishment of the transcriptome fluctuations along spermatogenesis, both for coding and for long non-coding transcripts (lncRNAs) [
94,
95]. Moreover, the purification of the L/Z population allowed for the first time the comparison of the RNAseq-derived transcriptomes of early meiotic prophase I (in which essential events such as homologous chromosome alignment and pairing occur) and medium/late meiotic prophase cells (in which crossing over takes place) [
94]. A comprehensive revision on the transcriptomics along male mouse gamete formation—and particularly of meiotic cells—is to be published elsewhere [
96].
Where certain cell types cannot be solely purified based on ploidy, different strategies have been employed such as the introduction of fluorescent labels. As an example, Zimmermann et al. (2015) generated
Sox9-
eGfp knock-in mice by the introduction of a cassette into the 3′ UTR of the endogenous
Sox9 gene [
97], which is expressed in Sertoli cells and encodes a transcription factor with a key role in testis development [
98]. This approach allowed labeling and sorting by FACS, GFP-positive Sertoli cells at five different time points corresponding to key stages of spermatogenesis. These were submitted to RNAseq for the characterization of the dynamic changes of Sertoli cell-coding and noncoding transcriptomes along the first wave of spermatogenesis, which showed the evolving roles of these cells along the process [
97]. In a different example, transgenic labeling in combination with FACS was used for the efficient identification and collection of spermatogonia from
Dazl (that encodes a germ cell-specific RNA-binding protein) knockout testes and WT controls, which were submitted to RNAseq aiming at the identification of mRNAs sensitive to
Dazl deletion. This study evidences a mechanism for DAZL–RNA binding and illustrates the role of DAZL as a master regulator of a post-transcriptional mRNA program essential for germ cell survival [
99].
So far in this review, no specific antibody-dependent identification and sorting of cell types has been considered. This possibility does exist and, in fact, the combination of FACS with antibody labeling has been a very common strategy for the isolation of cell types that are hardly distinguishable or not distinguishable at all in the cytometric profiles, such as specific populations of spermatogonia, or certain somatic cell types. One such example is a report by Harichandan et al. (2016), who simultaneously obtained different enriched cell populations from adult human testicular biopsies by multicolor staining with a combination of lineage-specific antibodies, followed by FACS sorting. Highly enriched spermatogonia and perivascular mesenchymal stromal cells were then submitted to RNAseq, for the analysis and comparison of their transcriptomic profiles [
100]. Actually, the different groups have employed diverse antibodies and strategies for sorting specific spermatogenic cell types that cannot (or are hard to) be purified based on their DNA content. A couple of examples on antibody-based sorting of spermatogonia that were then used for single-cell RNAseq (scRNAseq) are shown in the next section (see
Figure 9).
FACS has been also combined with other sorting technologies for further purification. For instance, Zhu and collaborators (2016) purified 2C, 4C and C cell populations from human testicular biopsies based on ploidy by FACS, followed by subjecting the 2C population to Magnetic-Activated Cell Sorting (MACS) with anti-CD90 antibody, for the enrichment in undifferentiated spermatogonia. The sorted cell populations were subsequently used for RNAseq profiling [
39].
Notably, FCM sorting has been also used as a tool to purify cell-type specific germ cell populations for proteomic analysis (e.g., [
101]), although we will not address the issue here.
5.2. FACS Contributions in Single Cell-Based Approaches
The advent of single-cell genomics has significantly expanded the possibilities to study the highly dynamic transcriptional programs underlying sperm production. Among all recent spermatogenesis molecular reports, single-cell-based ones have gained a prominent position. Single-cell RNAseq (scRNAseq) technology enables to profile the transcriptome of thousands of single cells in a population, thus eliminating the puzzling problem of testis heterogeneity. It allows detection and characterization of the existing heterogeneity at any given phase, as well as the RNA content of rare cell populations [
102,
103].
The collection of high-dimensional molecular data resulting from scRNAseq is most-commonly analyzed by a pseudotime algorithm to extract latent temporal information. Pseudotime ordering measures the relative progression of each of the cells and arranges them along a continuous path representing the spermatogenic process, thus capturing the continuity of spermatogenesis.
During the last three years, there have been various reports on scRNAseq for spermatogenesis profiling, employing either human testicular samples [
104,
105,
106,
107], or mouse testes, many of which have worked with unselected cells [
42,
43,
105,
108,
109,
110]. As this experimental design uses the cells coming directly from a testicular cell suspension, it allows for confidence on the relative proportions of cells and prevents undesired potential consequences of selection bias such as the eventual erroneous loss of rare or transitional cell types.
In various studies, FACS has complemented single-cell approaches. For instance, some of the above-mentioned reports on scRNAseq from unselected cells have also included the analysis of certain stage-specific cells purified by FACS, for more accurate cell-type assignment or for enrichment in certain cell types [
43,
105,
108,
110]. Such is the case of Green et al. (2018), who performed FACS-mediated 1n depletion and targeted enrichment in spermatogonia, Sertoli, and interstitial cells from adult mouse, to compensate for low abundance and allow a more comprehensive evaluation of these cell types found to be relatively underrepresented in their unselected dataset [
108].
Regarding methodological constraints, cell enrichments before scRNAseq profiling would have the disadvantage that altered cell proportions influence the statistics underlying pseudotime modeling. Anyway, the consequences of selection bias in the continuum pseudotime profile have proven moderate, at least in some cases. As an example, La et al. (2018) worked with selected
Plzf-mCherry+ undifferentiated spermatogonia (
Plzf encodes a transcriptional regulator that inhibits stem cell differentiation and is expressed in undifferentiated spermatogonia) and obtained a very similar pseudotime profile to others’ based on unselected spermatogonia [
111].
The use of specific markers has been indeed a common practice when the interest was on investigating spermatogonia and addressing developmental questions such as the origin of SSCs. Therefore, many scRNAseq studies have analyzed the transcriptomes of mouse prospermatogonia and/or spermatogonia selected by means of specific markers and isolated by FACS at certain time points after birth [
105,
112,
113,
114], during the embryonic phase [
115], or even from adult individuals [
105] (
Figure 9). Different laboratories have used a combination of transgenic fluorescent labels and antibodies, for FACS sorting (an example can be seen in
Figure 9A). The purification of SSCs will be specifically discussed later on. Importantly, for detailed lists on some of the most commonly used markers for the effective FACS sorting of SSCs from the testes of different mammals, recent review articles specifically addressing this topic, and tables therein, can be consulted [
116,
117]). Together, single-cell analyses have identified new subsets of spermatogonia and unveiled the dynamic nature of spermatogenic initiation.
FACS—based both on ploidy and on cell type-specific antibodies—has been also employed as a parallel strategy to random cell picking (unsorted) for sample collection. For instance, Wang et al. (2018) used both approaches to ensure the capture of all the testicular cell types from human donors with normal spermatogenesis and with NOA, before scRNAseq [
106]. Likewise, Jung et al. (2019), who introduced a novel model-based factor analysis method (sparse decomposition of arrays [SDA]) and applied it to the analysis of scRNAseq data from the testes of wild-type and mutant mice with gonadal defects, compared the expression profiles of cells from total testis dissociation, to those of testicular cells of known identity purified by FACS [
43]. Rather than clustering groups of cells (as in the rest of the reports discussed in this section), SDA identifies components comprising groups of genes that co-vary in expression and represents a single-cell transcriptome as a sum of those components. The analysis of the expression levels of known cell type markers and comparison to the FACS-sorted cells, enabled the resolution of 32 clusters into distinct subtypes of germ cells and somatic cell populations. This analytical strategy revealed a novel level of complexity, with multiple different components even within well-recognized meiotic stages such as the P stage [
43].
Thus far in this section, we have outlined reports based on the analysis of cells from specimens with unmanipulated, asynchronous spermatogenesis. In 2013, Hogarth and colleagues reported a novel synchronization method [
118], which has been recently applied upstream of scRNAseq studies [
40]. It consists of the administration of an inhibitor of RA synthesis (compound WIN18,446) to juvenile mice for 7 days, followed by a single dose of RA, which results in spermatogenic synchronization, with a dramatic simplification of testicular cell composition [
118]. Refined versions of this synchronization protocol have been developed, which yield predictable timing of germ cell development, allowing enrichment for precise developmental stages by simple timed collections [
40,
119]. The latter authors also devised FACS protocols to efficiently sort the synchronized germ cells. Their 3S method (synchronize, stage, and sort) combines in vivo simplification of the cellular composition of the testis and ex vivo cell sorting, thus achieving the obtainment of germ cell subpopulations from the undifferentiated spermatogonia through late meiotic prophase with ~90% purity and high yield [
108]. Compared to the unperturbed adult testis, synchronization would increase the percentage of germ cells at any given meiotic phase by a factor of at least 10 [
120], allowing rapid sorting [
119]. Moreover, despite the process of spermatogenesis being manipulated, synchronization would not interfere with normal germ cell biology and function, as synchronized animals have not shown overt differences in fertility or gene expression compared to control, unsynchronized testes [
118,
119].
Chen et al. (2018) used a combination of synchronization and transgenic labeling with
Vasa-dTomato (which is expressed in spermatogenic cells) and
Lin28-YFP (expressed in undifferentiated spermatogonia). This allowed the obtainment of 20 different spermatogenic cell types (including different types of spermatogonia, different stages of primary spermatocytes, secondary spermatocytes, and round spermatids at different steps of development) with over 90% purity, for scRNAseq profiling [
40]. Based on their single-cell dataset, they also identified a spermatid-specific surface marker (CD63) that was very effective in distinguishing round spermatids at different stages. This represents a highly relevant finding as it raises the possibility of employing this discriminative marker to accurately isolate round spermatids at different developmental steps by FACS, for downstream molecular studies on spermatid differentiation. This finding might also have eventual implications for assisted reproduction, as round spermatids are used for intracytoplasmic injections into oocytes (ROSI, ROund Spermatid Injections), and differences between the various spermatid stages concerning their developmental potential after injection might exist. In this regard, the authors performed intracytoplasmic ROSI using FACS-enriched synchronized spermatids at different stages and obtained significant differences in the ability to promote development beyond the stage of two-cell embryo, between the different sorted spermatid populations [
40]. In addition, the distinction and purification based on surface markers opens the possibility of sorting different spermatid stages from normal, asynchronous spermatogenesis.
5.3. FACS Contributions to Chromatin Conformation Studies
Mammalian genomes are organized into a highly dynamic chromatin structure, the regulation of which plays a critical role in biological processes involving DNA. During gamete generation, crucial DNA-based biological processes take place with associated chromatin remodeling. In meiosis, germ cells undergo programmed double strand breaks (DSBs) formation, homologous chromosome recognition, alignment, pairing, and recombination. Thus, highly dynamic chromosome movements occur along meiotic prophase I, generating peculiar configurations readily visualized at the light microscope level, such as the chromosome bouquet, i.e., a congregation of the telomeres at a nuclear envelope sector, originally described by Gelei a century ago [
121,
122,
123]. Particularly for male gamete formation, some unique chromatin features have been identified. During the P stage, sex chromosomes undergo a process termed meiotic sex chromosome inactivation (MSCI), by which the unsynapsed regions of X and Y chromosomes are subjected to male-specific transcription silencing [
124,
125]. Later on, during spermiogenesis, the sequential histone replacement, first by transition proteins, and then by protamines (see above), involves dramatic chromatin remodeling [
12,
126,
127]. In the few last years, there have been important advances in the understanding of chromatin accessibility, histone modifications, DNA methylomes, and three-dimensional (3D) chromatin structure, during mouse and/or human spermatogenesis [
41,
42,
44,
128,
129,
130,
131,
132,
133,
134]. Again, in many of these studies, FACS has played diverse important roles.
The contribution of epigenetic mechanisms to meiosis has been a long-relegated issue mostly due to inaccessibility to cell populations from all meiotic prophase I substages. In 2010, Getun et al. validated and refined Ho342-based FACS protocols, for the simultaneous purification of spermatogonia, pre-L, L/Z and P/D cell fractions from adult mice. The obtainment of all these cell populations (including L/Z, in which DSBs are generated), combined with micrococcal nuclease digestion and qPCR oligo-tiling assays, allowed to generate nucleosome occupancy maps, thus reporting that the cores of recombination hotspots have generally an open chromatin structure [
38]. Gaysinskaya and colleagues (2014) went one-step ahead in the sorting protocols, by developing an optimized Ho342-based FACS protocol that allowed finer purifications, enabling the attainment of separate populations for L and Z spermatocytes (instead of a joined L/Z population) with significant enrichment (60–80% and 75–90%, respectively) [
46]. This protocol, followed by genome-wide bisulfite sequencing, was applied to the study of DNA methylation in adult mouse spermatogonia, spermatocytes at all meiotic prophase I substages, and epididymal spermatozoa. They found that chromosomes exhibit a global transient reduction in DNA methylation in meiotic prophase, with a pronounced drop in pre-L followed by a progressive raise along the prophase stages, which suggests that key meiotic events occur in the context of dynamic changes in DNA methylation, perhaps facilitating them [
41].
FACS has been also used in combination with ChIP-seq for epigenetic studies. As an example, Lesch et al. (2013) performed ChIP-seq for the H3K4me3 mark (associated with active promoters) and the H3K27me3 mark (associated with facultatively repressed promoters) and RNA-seq, on flow-sorted male and female mice germ cells at different time points during embryogenesis. This analysis, which was extended to meiotic and postmeiotic male germ cells (purified by STA-PUT), identified a set of genes that is maintained in an epigenetically poised state across sexes and across developmental stages, including haploid postmeiotic cells [
135]. On the other hand, Ernst et al. (2019) combined FACS-sorted spermatocyte and spermatid cell populations from juvenile mice at different ages, with Cleavage Under Targets and Release Using Nuclease (CUT&RUN) [
42], an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease, releases specific protein–DNA complexes into the supernatant for paired-end DNA sequencing [
136]. They characterized the epigenetic changes underlying X chromosome re-activation after MSCI and found a set of genes strongly enriched in the repressive mark H3K9me3 in spermatocytes, which then undergo extensive post-meiotic chromatin remodeling thus acquiring an active chromatin state in round spermatids [
42].
FACS-sorted testicular cell populations have been also employed in Hi-C studies (high-throughput genome-wide chromosome conformation capture sequencing), aiming at the establishment of the 3D organization of the genome in germ cells. Vara and colleagues (2019) addressed the issue by integrating Hi-C, RNAseq, and ChIPseq of CCCTC-binding factor (CTCF) and meiotic cohesins [
44]. They implemented a reproducible FCM protocol to isolate enriched male mouse germ cell populations representing different stages of spermatogenesis: premeiotic spermatogonia, meiotic (L/Z, and P/D), and post-meiotic cells (round spermatids and sperm) (
Figure 10). Cell suspensions were stained with Ho342, and an “in solution” immunofluorescence using primary antibodies against proteins DMC1 (a meiotic recombinase) and SYCP3 (a SC component) was performed to clearly discriminate and sort L/Z (DMC1+/SYCP3+) from P/D spermatocytes (DMC1-/SYCP3+). This sorting strategy represents an effective way to prevent cross contamination between L/Z and P/D cells (see
Figure 10A), as well as to get rid of eventual contaminating G2 spermatogonia. The in-parallel application of multiple techniques on the various cell populations classified by FACS allowed the authors to find a correlation between gene expression, cohesin occupancy, and local insulation during chromatin reorganization along spermatogenesis. Among other interesting results, they observed a major structural re-organization, with dynamic changes in topological associating domains (TADs) and chromatin compartmentalization along spermatogenesis; a possible novel role for meiotic cohesins in genome organization and function during meiotic prophase I and spermiogenesis; and a differential chromatin pattern and cohesin loading in the sex chromosomes, which most likely reflects MSCI [
44].
In another recent report, Patel et al. (2019) performed Hi-C in both early prophase (Z) and late prophase (late-P/D) spermatocytes isolated by FACS from mice with synchronized spermatogenesis by the WIN18,446/RA method, aiming to study chromatin organization dynamics during the recombination process [
134]. By working with synchronized testes, they obtained FCM profiles with only a few well-separated and densely populated 4C regions, facilitating their classification (
Figure 11). Besides providing information about the dynamic changes in TADs and chromatin compartmentalization during mammalian spermatogenesis, they observed that DSBs and crossovers show a strong bias toward the gene-dense compartment, suggesting a role for chromatin state in meiotic recombination. Concerning the X chromosome, in coincidence with the above-mentioned report and also with others [
132,
133], their results point to a distinct higher-order chromatin organization during MSCI [
134].
5.4. Purification of Spermatogenic Cells for In Vitro Culture Developments
In vitro recapitulation of the spermatogenic process has been a precious goal among reproductive biologists for at least six decades. It represents a high challenge given the complexity of germ cell proliferation and differentiation, which require precise niche conditions and signals, including cellular interactions within the seminiferous ephitelium, and the presence of growth factors and hormones [
18,
137,
138,
139,
140]. The availability of an effective spermatogenic in vitro system would facilitate, among others, studies on the requirements of the process in a controlled in vitro environment; research that is difficult and/or unethical to perform directly in vivo; studies on the molecular mechanisms of pathologies such as testicular cancer or male infertility; fertility restoration or preservation, by the production of haploid male germ cells from undifferentiated germ cells isolated from infertile adult patients, or from pre-pubertal cancer patients before the application of gonadotoxic treatments, respectively [
17,
138,
139,
141]. Different strategies and advances for the development of in vitro spermatogenesis have been devised in the last years and reviewed elsewhere [
18,
117,
139,
140,
142] and are far beyond the scope of this revision.
As SSCs are responsible for the continuous production of spermatogonia that sustain spermatogenesis [
143], the generation of in vitro culture systems to efficiently maintain and expand SSCs is fundamental for progress towards the above-mentioned goals [
117,
144]. The first step for the development of an SSCs culture system is the isolation of these cells, which is challenging as for instance in mouse testicles they are extremely scarce, representing only ~0.03% of total germ cells [
145]. Since isolation of SSCs requires their distinction from other spermatogonial differentiation stages, both MACS and FACS have been employed in combination with specific biochemical markers for their sorting and culture. MACS can isolate highly purified cells from a complex cell mixture, based on cell surface antigen specificity. However, it cannot differentiate some subpopulations with distinct biological characteristics. In this regard, FACS presents important advantages for the obtainment of high purity SSCs, as it provides morphological data of cells, allows the simultaneous detection of multiple surface markers, and is informative on eventual quantitative differences of biochemical markers between cellular subpopulations (e.g., the distinction of Thy1
dim and Thy1
bright subpopulations within Thy-1
+ populations, in human testicular cells) [
146]. It is generally agreed that satisfactory results for FACS sorting of SSCs requires multiple markers. The advances in the molecular knowledge of testicular cells have allowed the identification of different molecular markers, which in turn has generated new tools for FACS purification. Accordingly, as above mentioned, recent revisions on markers available to distinguish and sort SSCs in different species have been published [
116,
117].
Regenerative capacity is the feature that better defines stem cells, and research on this aspect requires functional assays. Transplantation from mice donor testes into the testes of recipient mice has proven useful in assessing the regenerative capacity of different subsets of SSCs [
117,
138,
141,
145]. Again, FCM based on specific molecular markers has been a highly valuable tool in the discrimination between different stem cell subsets [
147], to identify those with regenerative capacity that will lead to spermatogenic colonies within recipient testes.
As the field of in vitro spermatogenesis is a highly prolific research area with enormous clinical implications, so far the number of published reports is huge. Here we have selected only a few examples, to illustrate the wide variety of contributions of FCM to the subject.
In adult mice, it has been established that less than 10% of A
s spermatogonia have transplantation capability [
148]. More recently, some have concluded that transplant activity is almost exclusively contained within a fraction of A
s marked by ID4 (a transcriptional repressor with a critical role in the balance regulation between self-renewal and differentiation of SSCs) expression, thus supporting a revised A
s model in which stem cell activity is limited to a subset of A
s [
149,
150]. Law et al. (2019) analyzed the kinetics and mechanisms underneath the appearance, during neonatal life, of the SSC foundational pool derived from prospermatogonial precursors in mouse, by employing a multi-transgenic reporter mouse model, FACS classification, scRNAseq, and transplantation analyses to define the SSC trajectory from prospermatogonia. Their results suggest that SSC fate is pre-programmed within a subset of fetal prospermatogonia, before the establishment of the foundational pool during early neonatal development [
151].
An alternative to the revised A
s model was proposed by Nakagawa and colleagues a decade ago, introducing the existence of multiple and reversible paths from stem cells to differentiation [
152]. This model conceived stem cell potential as a dynamic property shared by most undifferentiated type A spermatogonia (A
undiff) and proposed that gene expression would dictate fate tendency. Recent evidence supporting this model has started to emerge. La et al. (2018), by using the
Plzf reporter system for A
undiff FACS purification from mouse, uncovered an unappreciated population within the self-renewing A
undiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1, with potent stem cell capacity [
111]. Interestingly, upon transplantation and culture, they demonstrated the existence of dynamic cell states for A
undiff, which interconvert between PDX1
+ and PDX1
− states [
111].
Velte et al. (2019) addressed the issue of how spermatogonial response to RA is regulated in mouse at the molecular level, and the importance of the microenvironment, by using in-parallel in vivo and in vitro approaches [
153]. To obtain spermatogonia, they employed
Id4-eGfp mice and isolated EGFP
+ spermatogonia by FACS, to discriminate spermatogonia differing in their regenerative capacity (ID4-EGFP
bright spermatogonia are highly enriched in SSCs, while ID4-EGFP
dim and ID4-EGFP
− have no regenerative capacity and correspond to progenitor and differentiating spermatogonia, respectively [
149,
154]). By examining the intrinsic RA responsiveness of isolated spermatogonia maintained in vitro without somatic cells, they observed similar responsiveness in vivo and in vitro, thus indicating that differential RA responsiveness is an intrinsic feature of developing male germ cells, with minimal dependence on an in vivo microenvironment [
153].
Others have addressed studies on human SSCs. As an example, Sohni and collaborators (2019), by employing human adult testicular biopsies and neonatal testes for scRNAseq, were able to detect four different clusters of undifferentiated spermatogonia. One of them exhibited the characteristics of highly enriched SSCs, for which two molecular markers were identified (LPPR3 and TSPAN33) and used in FACS purifications and RT-qPCR analyses. They also identified genes expressed by testicular somatic cells, including those encoding cell–cell signaling factors, which may prove useful in developing cocktails with factors to favor the expansion of human SSCs in vitro, for eventual in vivo clinical applications [
155].
Despite efficient SSC purification and transplantation being a promising option for male fertility preservation in pre-pubertal cancer patients, the safety of such application is controversial, given the risk of introducing malignant cells along with the SSCs. This issue has been addressed by different laboratories. For instance, Tian et al. (2019) compared the relative safety of three different purification methods—Percoll density gradient, MACS and FACS—for application of SSC transplantation, in testes of leukemia model mice. Interestingly, after 16 weeks, no malignant cells were found in mice transplanted with MACS- or FACS-purified SSCs, which suggests that these methods could be safely applied without concomitant tumor implantation [
156] (
Figure 12).
Finally, we want to acknowledge the important role FCM has also played during the establishment of culture systems, for the analysis and quantitation of the SSCs in culture at different time points employing specific markers (e.g., [
157]).
