Sex,the Underestimated Potential Determining Factor in Brain Tissue Repair Strategy

Introduction

T he development of stem cell-based therapy has triggered a lot of hype as a strategy for brain repair. However, despite intensive preclinical research, clinical translation seems to be delayed since the first clinical trial in 1996 [1]. Conversion into novel therapies still faces many technical hurdles of neural stem cell (NSC) expansion, maintenance, assessment of stemness, capacity for neurogenesis, and storage. In addition, there is still no consensus in the field as to which stem cells (embryonic, adult or induced progenitor cells) should be developed for a given therapeutic application. In addition, the anecdotically favorable results obtained by the few clinical trials conducted in the past 5 years [2 –8] show that we lack critical knowledge on stem cells biology and would benefit from a return to fundamental work. The goal of the present review is to discuss the therapeutic relevance of the largely under-explored sex- and age-based differences in the capacity of NSCs to engage in neurogenesis. Indeed, beyond understanding the physiology and biochemistry of aging NSCs, for both sexes, the overall objective is to discuss the potential foundation for future studies aimed at tailoring NSC transplantation strategies for brain repair as a function of sex/age, as well as to consider sex- and age-specific pharmacological approaches toward the development of neurogenesis-inducing treatments.

NSCs Sexual Dimorphism

The brain is a main target of gonadal steroid hormones and contains many of the steroid metabolizing enzymes. The effect of gonadal steroids on brain development and maturation has been extensively described [9,10]. The vast literature on the subject has introduced the common belief that gonadal steroids are the only effectors of the brain sexual differentiation [11 –13], and have overshadowed other potential key elements. Although the importance of sex steroids in brain maturation is unquestionable, the dogma of the gonadal origin of somatic differentiation[14], including neuronal cells, usually implies that male and female cells and, therefore, stem cells are functionally identical unless gonadal steroids act on them in a sex-dependent manner. The human Y chromosome encodes 27 different proteins [15], 8 of which are expressed in the male brain. It is, therefore, highly conceivable that these male proteins could have a male-specific effect on the brain, independent of any gonadal hormone influence [16,17]. Further, female cells contain an X chromosome that received a paternal genomic imprint, whereas male cells do not, and this fact is likely to contribute to intrinsic differences between male and female cells. De Vries and colleagues [18] generated mice in which the testis-determining gene sry was deleted from the Y chromosome and subsequently inserted onto an autosome. The testis of these mice were developed independently of the complement of X or Y chromosome. Although most of the sexual dimorphism correlated with the presence of testis or ovary (and was, therefore, associated with gonadal hormones), XY mice (with testes or ovaries) were found to be more masculine than the XX mice (with testes or ovaries). In particular, the density of vasopressin-immunoreactive fibers in the lateral septum was found to be more masculine, suggesting that sex chromosome genes play a significant but largely underestimated role in the development of a sex difference in the brain. These results also suggest that female and male NSCs should not be regarded as equivalent and should not be a priori considered to necessarily react in the same manner to a specific environment or pharmacological agent. Moreover, there are no data to support the a priori consideration that transplanting young female NSCs in an old female brain would result in the same neural differentiation and functional recovery as transplanting young male NSCs in an old male brain. Likewise, there is no evidence to a priori consider that male and female NSC neurogenic properties would evolve in a sex-independent manner throughout development and aging. Sexual dimorphism has already been described in various functional aspects of several types of stem cells [19 –25] and certainly supports this hypothesis. In particular, we recently reported a sexual dimorphism in the neurogenic capacity of rat [26,27] NSCs, and similar observations have been made by others in primate [25] NSCs. Considering the dramatic and sex-specific hormonal changes occurring throughout development and aging, one might expect a sex- and sex-through-aging-specific environment to be a prerequisite for successful neurogenesis.

We have recently published compelling evidence that the estrogen receptor ERα is differentially expressed in male and female NSCs. We have found that NSCs isolated from 3 month-old rats display sexual dimorphism in the expression of both estrogen receptor subtypes [26]. Specifically, young male NSCs contain one third of the ERα levels, whereas ERβ levels were thrice greater than those expressed by the female NSCs of the same age. Moreover, our data demonstrated that the ERα/ERβ ratio was close to 1 in the male but 10-fold higher in the female NSCs [26]. These results are supported by previous data showing that steroid receptor expression in the fish inner ear varies between sexes [28]. Such sexual dimorphism has been previously described in mature neurons of various brain structures [29,30] and has been shown to have a role in the differentiation of sexual behavior and gender identity [31], with ERα being primarily involved in masculinization, whereas ERβ is primarily involved in defeminization [32,33]. Although it may sound evident, starting from birth and throughout life, sex hormones physiology and homeostasis are different between men and women. Therefore, one has to bear in mind that transplanting the “wrong” type of NSC to the “wrong” patient may have detrimental consequences. Indeed, consequences in clinics may run from a lack of recovery to partial or inadequate recovery. Dissimilarity between NSCs and the recipient tissue may cause these undesirable effects. Interestingly, in the same manner, NSCs isolated from 20 month old male and female rats displayed a dramatic increase in ERα and ERβ expression that was equivalent in both sexes, suggesting that male and female NSCs are not equal before aging. The effect of estrogens on neurogenesis has been extensively studied, and it is commonly agreed that estrogens simultaneously promote NSCs proliferation and differentiation [34 –39]. Despite mounting evidence on the estrogenic aspect of neurogenesis, the respective roles of ERα and ERβ in this process still remain to be fully characterized. Considering the currently known role of estrogens in NSCs proliferation and differentiation [34,36,39,40 –43], the difference in ERα and ERβ expression between male and female NSCs [26] that we observed likely advocates for a sex-based intrinsic difference in the regulation of neurogenesis. In addition, a recent report established that ERα genotype is responsible for the inter-individual variability of responses to estrogen and testosterone in mesenchymal stem cell-derived osteoblasts [44], and that estradiol altered neurogenesis in female, but not male rats [45]. Further, we also showed that males NSCs expressed a dramatically higher level of CYP19 than female NSCs [27] in which CYP19 was barely detected. Such a sexual dimorphism suggests a capacity for male NSCs, unlike female NSCs, to metabolize testosterone and, in turn, to produce estradiol, therefore providing male NSCs with the ability to alter their local environment and modulate endogenous neurogenesis in a different manner than female NSCs may do. Interestingly, the existence of autocrine control loop in NSCs has already been described for the kinin/kallicrein system [46] and in the androgenic apparatus of human bone marrow stromal cells [47]. Remarkably, the sex-based differences unveiled in vitro translated in vivo as recently reported [48]. Indeed, the outcome of NSCs transplantation in brain was shown to tightly depend on the sex of the donor and the sex of the recipient. Interestingly, in some cases, cross-sex grafting provided better cell survival results than same-sex transplantation.

NSCs Sex-Through-Aging Related Differences

It is commonly understood that age-associated alterations in the brain play an important role in the decline of neurogenesis reported in aging [25,49]. However, very little work has been done in defining the age-related alteration of NSCs intrinsic differentiation ability. Old mice have been shown to house less NSCs in the subventricular zone (SVZ) compared with young ones, and a similar reduction has been reported in the number of NSCs maintained as neurospheres and recovered in culture in vitro [50]. A decrease of NSC proliferation has been described in the hippocampus of old rats [51] but not in the SVZ of the same animal. In contrast, NSCs seem to undergo a spatial redistribution within the SVZ and a delay in the migratory process rather than a decrease of the proliferation rate [52]. However, although some studies showed that NSCs from old animals retain some or all of their neurogenic properties [53,54], to date, studies that have systematically explored age-based differences of NSCs neurogenic properties in terms of neuronal phenotype and protein marker expression are extremely scarce [25 –27,55 –57]. Surprisingly, the survival rate of the transplanted cells was not necessarily better with NSCs isolated from young animals compared with the ones isolated from old rats [48]. Indeed, transplantation outcome also depended on the sex of the recipient [48]. Such a result provocatively raises the question of the rejuvenation process that NSCs isolated from old individuals may undergo after being transplanted in a younger environment as suggested by 2 recent works [58,59]. From the perspective of stem cell grafting into the brain, it is critical to have a good understanding of the neuronal fate specificity and the maturation level reached by the engrafted NSCs as a factor of the NSCs donor age. In addition, in agreement with our previous results showing that NSCs age differently depending on their sex, it appears that sex cannot be dissociated from age as a determining factor of NSCs capacity to lead to functional recovery after transplantation. Further, we foresee that NSC differentiation induced by neurogenic agents in vitro and in vivo will be an important part of brain repair procedures, as brain repair therapy and optimal neuronal function restoration will likely require both exogenous NSC grafting and pharmacological stimulation of the endogenous neurogenesis. However, nothing is currently known about the effect of aging on male and female NSC sensitivity to pharmacologically induced differentiation, and we, therefore, strongly believe that acquiring such knowledge is critical for the development of neurogenic treatments.

Conclusion

Stem cells hold a lot of promises for brain repair, tissue regeneration, and function recovery. However, translation in clinics seems to be hampered by several hurdles mainly related to our lack of knowledge in NSC physiology. Moreover, the very low success rate encountered by the few clinical trials conducted to date certainly advocates for a return to a more fundamental bench work. In particular, mechanisms and factors that pilot NSC survival, migration, and differentiation after transplantation need to be unveiled. Among these factors, recent works conducted in our laboratory and by others suggest that stem cell sex may be critical to the transplantation outcome and tissue regeneration, therefore warranting further investigation. In this article, we discussed NSCs intrinsic sexual dimorphism that may have a significant translated impact in clinics. In particular, we emphasize how differential expression in steroid receptors and androgen metabolizing enzyme may account for differential outcome of NSCs transplantation. Such data may preach against the orthodoxy in place, but taking NSCs sex into consideration may hold part of the answer to the question “What NSC for whom?” and may improve the translation of sound research into novel brain repair strategies.