Stem cell/cellular interventions in human spinal cord injury: Is it time to move from guidelines to regulations and legislations? Literature review and Spinal Cord Society position statement.

PURPOSE: In preclinical studies, many stem cell/cellular interventions demonstrated robust regeneration and/or repair in case of SCI and were considered a promising therapeutic candidate. However, data from clinical studies are not robust. Despite lack of substantial evidence for the efficacy of these interventions in spinal cord injury (SCI), many clinics around the world offer them as "therapy." These "clinics" claim efficacy through patient testimonials and self-advertisement without any scientific evidence to validate their claims. Thus, SCS established a panel of experts to review published preclinical studies, clinical studies and current global guidelines/regulations on usage of cellular transplants and make recommendations for their clinical use. METHODS: The literature review and draft position statement was compiled and circulated among the panel and relevant suggestions incorporated to reach consensus. This was discussed and finalized in an open forum during the SCS Annual Meeting, ISSICON. RESULTS: Preclinical evidence suggests safety and clinical potency of cellular interventions after SCI. However, evidence from clinical studies consisted of mostly case reports or uncontrolled case series/studies. Data from animal studies cannot be generalized to human SCI with regard to toxicity prediction after auto/allograft transplantation. CONCLUSIONS: Currently, cellular/stem cell transplantation for human SCI is experimental and needs to be tested through a valid clinical trial program. It is not ethical to provide unproven transplantation as therapy with commercial implications. To stop the malpractice of marketing such "unproven therapies" to a vulnerable population, it is crucial that all countries unite to form common, well-defined regulations/legislation on their use in SCI. These slides can be retrieved from Electronic Supplementary Material.

Defective Wnt3 expression by testicular Sertoli cells compromise male fertility.

Testicular Sertoli cells make a niche for the division and differentiation of germ cells. Sertoli cells respond to increased follicle-stimulating hormone (FSH) and testosterone (T) levels at the onset of puberty by producing paracrine factors which affect germ cells and trigger robust onset of spermatogenesis. Such paracrine support to germ cells is absent during infancy, despite Sertoli cells being exposed to high FSH and T within the infant testis. This situation is similar to certain cases of male idiopathic infertility where post-pubertal Sertoli cells fail to support germ cell division and differentiation in spite of endogenous or exogenous hormonal support. Defective Sertoli cells in such individuals may fail to express the full complement of their paracrine repertoire. Identification and supplementation with such factors may overcome Sertoli cells deficiencies and help trigger quantitatively and qualitatively normal differentiation of germ cells. To this end, we compared the transcriptome of FSH- and T-treated infant and pubertal monkey Sertoli cells by DNA microarray. Expression of Wnt3, a morphogen of the Wnt/ß-catenin pathway, was higher in pubertal Sertoli cells relative to infant Sertoli cells. Transgenic mice were generated by us in which Wnt3 expression was curtailed specifically in post-pubertal Sertoli cells by shRNA. Subfertility and oligozoospermia were noticed in such animals with low Wnt3 expression in post-pubertal Sertoli cells along with diminished expression of Connexin43, a gap-junctional molecule essential for germ cell development. We report that the FSH- and T-targetedf Wnt3 governs Sertoli cell-mediated regulation of spermatogenesis and hence is crucial for fertility.

Clinical translation of stem cell based interventions for spinal cord injury - Are we there yet?

Recent advances in basic science in research related to spinal cord injury (SCI) and regeneration have led to a variety of novel experimental therapeutics designed to promote functionally effective axonal regrowth and sprouting. Stem cell and other cellular interventions have gained lot of attention due to their immense potential of regeneration. These interventions have been tested for their efficacy in case of SCI both at the pre-clinical and clinical level. In this review we critically discuss the published literature on the cellular interventions for SCI and their clinical applications with respect to the strength of evidence established by these studies. The need to curb unethical practice of offering unproven stem cell "therapies" for SCI at a global level is also discussed.

Stem cell therapy in spinal trauma: Does it have scientific validity?

Stem cell-based interventions aim to use special regenerative cells (stem cells) to facilitate neuronal function beyond the site of the injury. Many studies involving animal models of spinal cord injury (SCI) suggest that certain stem cell-based therapies may restore function after SCI. Currently, in case of spinal cord injuries, new discoveries with clinical implications have been continuously made in basic stem cell research, and stem cell-based approaches are advancing rapidly toward application in patients. There is a huge base of preclinical evidence in vitro and in animal models which suggests the safety and clinical efficacy of cellular therapies after SCI. Despite this, data from clinical studies is not very encouraging and at times confounding. Here, we have attempted to cover preclinical and clinical evidence base dealing with safety, feasibility and efficacy of cell based interventions after SCI. The limitations of preclinical data and the reasons underlying its failure to translate in a clinical setting are also discussed. Based on the evidence base, it is suggested that a multifactorial approach is required to address this situation. Need for standardized, stringently designed multi-centric clinical trials for obtaining validated proof of evidence is also highlighted.

Low levels of Gαs and Ric8b in testicular sertoli cells may underlie restricted FSH action during infancy in primates.

FSH acts via testicular Sertoli cells (Sc) bearing FSH receptor (FSH-R) for regulating male fertility. Despite an adult-like FSH milieu in infant boys and monkeys, spermatogenesis is not initiated until the onset of puberty. We used infant and pubertal monkey Sc to reveal the molecular basis underlying developmental differences of FSH-R signaling in them. Unlike pubertal Sc, increasing doses of FSH failed to augment cAMP production by infant Sc. The expression of Gαs subunit and Ric8b, which collectively activate adenylyl cyclase (AC) for augmenting cAMP production and gene transcription, were significantly low in infant Sc. However, forskolin, which acts directly on AC bypassing FSH-R, augmented cAMP production and gene transcription uniformly in both infant and pubertal Sc. FSH-induced Gαs mRNA expression was higher in pubertal Sc. However, Gαi-2 expression was down-regulated by FSH in pubertal Sc, unlike infant Sc. FSH failed, but forskolin or 8-Bromoadenosine 3',5'-cyclic monophosphate treatment to infant Sc significantly augmented the expression of transferrin, androgen binding protein, inhibin-ß-B, stem cell factor, and glial-derived neurotropic factor, which are usually up-regulated by FSH in pubertal Sc during spermatogenic onset. This suggested that lack of FSH mediated down-regulation of Gαi-2 expression and limited expression of Gαs subunit as well as Ric8b may underlie limited FSH responsiveness of Sc during infancy. This study also divulged that intracellular signaling events downstream of FSH-R are in place and can be activated exogenously in infant Sc. Additionally, this information may help in the proper diagnosis and treatment of infertile individuals having abnormal G protein-coupled FSH-R.

Dickkopf homolog 3 (DKK3) plays a crucial role upstream of WNT/ß-CATENIN signaling for Sertoli cell mediated regulation of spermatogenesis.

Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4-6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/ß-CATENIN activity. Elevated ß-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/ß-CATENIN modulation.

A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats.

FSH and Testosterone (T) regulate spermatogenesis via testicular Sertoli cells (Sc), which bear receptors for these hormones. Despite sufficient circulating levels of FSH and T postnatally, predominant appearance of spermatogonia B and spermatocytes is not discernible until 11 and 18 days of postnatal age, respectively, in rat testes. In an attempt to explore the underlying causes, we cultured Sc from neonatal (5- and 9-day-old) and prepubertal (12- and 19-day-old) rat testes and compared the status of FSH receptor (FSH-R) and androgen receptor (AR) signaling. Protein and mRNA levels of FSH-R and AR remained uniform in cultured Sc from all age groups. Androgen binding ability of AR was similar, and T-induced nuclear localization of AR was discernible in Sc from all age groups. Binding of FSH to FSH-R, subsequent production of cAMP, and mRNA of stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF), known to be essential for the robust differentiation of repopulating spermatogonia, were significantly augmented in prepubertal Sc compared with those in neonatal Sc. However, treatment of neonatal Sc with cholera toxin or forskolin, which stimulate cAMP production bypassing FSH-R, demonstrated a concomitant rise in SCF and GDNF mRNA expression, which was similar to the FSH-mediated rise observed in prepubertal Sc. These observations suggested that, during prepubertal Sc maturation, the ability of FSH-R to respond to FSH is significantly augmented and is associated with the robust differentiation of repopulating spermatogonia, and such a switch in Sc from FSH-resistant to FSH-responsive mode during prepubertal development may underlie the initiation of robust spermatogenesis.

Insufficient androgen and FSH signaling may be responsible for the azoospermia of the infantile primate testes despite exposure to an adult-like hormonal milieu.

BACKGROUND: In humans, as well as in other higher primates, the infantile testis is exposed to an adult-like hormonal milieu, but spermatogenesis is not initiated at this stage of primate development. In the present study, we examined the molecular basis of this intriguing infertile state of the primate testis. METHODS: The integrity of androgen receptor (AR) and FSH receptor (FSHR) signaling pathways in primary cultures of Sertoli cells (Scs) harvested from azoospermic infant and spermatogenic pubertal monkey testes were investigated under identical in vitro hormonal conditions. In order to synchronously harvest Scs from early pubertal testis, the activation of testicular puberty was timed experimentally by prematurely initiating gonadotrophin secretion in juvenile animals with an intermittent infusion of gonadotrophin-releasing hormone. RESULTS: While qRT-PCR demonstrated that AR and FSHR mRNA expression in Scs from infant and pubertal testes were comparable, androgen-binding and FSH-mediated cAMP production by infant Scs was extremely low. Compromised AR and FSHR signaling in infant Scs was further supported by the finding that testosterone (T) and FSH failed to augment the expression of the T responsive gene, claudin 11, and the FSH responsive genes, inhibin-ßB, stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF) in Scs harvested at this stage of development. CONCLUSION: These results indicate that compromised AR and FSHR signaling pathways in Scs underlie the inability of the infant primate testis to respond to an endogenous hormonal milieu that later in development, at the time puberty, stimulates the initiation of spermatogenesis. This finding may have relevance to some forms of idiopathic infertility in men.

A method for rapid generation of transgenic animals to evaluate testis genes during sexual maturation.

In certain forms of idiopathic infertility, there is failure of follicle stimulating hormone (FSH) and testosterone (T) to initiate spermatogenesis despite the presence of Sertoli cells and germ cells in the testis. In postnatal rats (up to 11 days of age) and infant monkeys (3-4 months old), robust division and differentiation of spermatogonial stem cells is not discerned, even though serum levels of FSH and T are similar to those found during adulthood. Lack of spermatogenesis together with normal hormone levels is a situation similar to that found in certain categories of male infertility. To investigate this intriguing situation, Sertoli cells were cultured from infant and pubertal rats and monkeys and differential gene expression by testicular Sertoli cells was evaluated by DNA microarray using the Agilent microarray system. To determine the role of candidate genes in regulation of spermatogenesis, transgenic animals over-expressing these genes must be generated. However, present techniques for generation of transgenic animals have limited utility for production of several transgenic animals within a short period of time. Therefore, we have developed a technique for making transgenic animals by the testicular route which is less labor intensive and less time consuming. This technique is also ethically superior since fewer mice are required than in existing alternative methods of transgenesis.

Human embryonic stem cells: preclinical perspectives.

Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

Endotoxin-induced silencing of mesoderm induction and functional differentiation: role of HMGB1 in pluripotency and infection.

OBJECTIVES: Mechanisms underpinning Gram-negative bacterial vaginosis-induced birth anomalies are obscure. Ethical issues limit such studies on peri-implantation-stage human embryos. Here we have used embryoid bodies (EBs) as an in vitro model to examine the effect of Gram-negative bacterial endotoxins/lipopolysaccharides (LPS) on the faithful induction of germ lineages during embryogenesis. The role of LPS-inducible cytokine and pluripotency-related DNA-binding protein HMGB1 was also studied in these EBs. METHODS: EBs derived from the human embryonic stem cell line HUES9 were exposed to 12.5 pg/ml of LPS for 48 h. The expression profile of the ectoderm, endoderm, mesoderm and trophectoderm lineage markers, such as beta III-tubulin, GATA4, BMP2, Brachury and beta-hCG, were studied, by RT-PCR and immunofluorescence. Inhibition of mesoderm induction was confirmed by RT-PCR analysis for hANP, cTnT, ABCG2, GATA2, BMP4 and HAND1. Osteoblast differentiation was induced in the EBs, and confirmed by von Kosa and Alizarin red staining. A comet assay was also carried out to assess the degree of apoptosis in these EBs. RESULTS AND CONCLUSIONS: We found that the LPS-treated EBs were selectively silenced for mesoderm markers and failed to differentiate into functional osteoblasts. HMGB1 expression was absent in the normal EBs and was found to be localized in the cytoplasm of the LPS-treated EBs. Overall, our data indicate that endotoxin-induced HMGB1 expression in the peri-implantation-stage embryos can bring about severe birth defects of, for example, the bone and heart. This study also indicates that HMGB1 could be involved in maintenance of pluripotency in the human embryonic stem cells by impeding their differentiation.

Follicle-stimulating hormone-independent functions of primate Sertoli cells: potential implications in the diagnosis and management of male infertility.

CONTEXT: FSH is known to augment the production of essential germ cell (Gc) survival factors, lactate and estradiol, by Sertoli cells (Sc) of 18-d-old pubertal rats. However, the failure of gonadotropin and androgen treatment to initiate spermatogenesis in testis of some infertile men bearing Sc and Gc is intriguing. The role of FSH in regulation of lactate and estradiol production by primate Sc is currently unknown. OBJECTIVE: The objective of the study was to determine the role of FSH in regulating lactate and estradiol production by primate Sc. METHODS: Gc differentiation was initiated in male juvenile rhesus monkeys by pulsatile administration of GnRH for 4-5 wk. Sc from these pseudopubertal monkeys and pubertal rats were cultured. Production of lactate and estradiol in response to FSH and 8-bromoadenosine-cAMP was evaluated. Inhibin-betaB expression, cAMP production, and cell proliferation were also assayed. RESULTS: Unlike Sc from pubertal rats, Sc from pseudopubertal monkeys constitutively aromatized testosterone to estradiol and produced large amounts of lactate without FSH stimulation. Increasing doses of recombinant monkey FSH or 8-bromoadenosine-cAMP failed to augment lactate production, although they significantly augmented proliferation of Sc. Production of cAMP and expression of inhibin-betaB mRNA were also remarkably augmented by recombinant monkey FSH. CONCLUSIONS: These results suggest that lactate and estradiol production by monkey Sc is not governed by FSH, as previously thought based on studies of rat Sc. Thus, in a clinical situation, assessment of such gonadotropin-independent functions of Sc may be obligatory for the diagnosis and management of certain forms of idiopathic male infertility.