Searching for a Cell-Based Therapeutic Tool for Haemophilia A within the Embryonic/Foetal Liver and the Aorta-Gonads-Mesonephros Region.

The development of new strategies based on cell therapy approaches to correct haemophilia A (HA) requires further insights into new cell populations capable of producing coagulation factor VIII (FVIII) and presenting stable engraftment potential. The major producers of FVIII in the adult are liver sinusoidal endothelial cells (LSECs) and in a lesser degree bone marrow-derived cells, both of which have been shown to ameliorate the bleeding phenotype in adult HA mice after transplantation. We have previously shown that cells from the foetal liver (FL) and the aorta-gonads-mesonephros (AGM) haematopoietic locations possess higher LSEC engraftment potential in newborn mice compared with adult-derived LSECs, constituting likely therapeutic targets for the treatment of HA in neonates. However, less is known about the production of FVIII in embryonic locations. Quantitative polymerase chain reaction and Western blot analysis were performed to assess the relative level of FVIII production in different embryonic tissues and at various developmental stages, identifying the FL and AGM region from day 12 (E12) as prominent sources of FVIII. Furthermore, FL-derived VE-cad+CD45-Lyve1+/- endothelial/endothelial progenitor cells, presenting vascular engraftment potential, produced high levels of F8 ribonucleic acid compared with CD45+ blood progenitors or Dlk1+ hepatoblasts. In addition, we show that the E11 AGM explant cultures expanded cells with LSEC repopulation activity, instrumental to further understand signals for in vitro generation of LSECs. Taking into account the capacity for FVIII expression, culture expansion and newborn engraftment potential, these results support the use of cells with foetal characteristics for correction of FVIII deficiency in young individuals.

Stem cells survive oncotherapy & can regenerate non-functional gonads: A paradigm shift for oncofertility.

A large proportion of patients who survive cancer are rendered infertile as an unwanted side effect of oncotherapy. Currently accepted approaches for fertility preservation involve banking eggs/sperm/embryos or ovarian/testicular tissue before oncotherapy for future use. Such approaches are invasive, expensive, technically challenging and depend on assisted reproductive technologies (ART). Establishing a gonadal tissue bank (for cancer patients) is also fraught with ethical, legal and safety issues. Most importantly, patients who find it difficult to meet expenses towards cancer treatment will find it difficult to meet expenses towards gonadal tissue banking and ART to achieve parenthood later on. In this review an alternative strategy to regenerate non-functional gonads in cancer survivors by targeting endogenous stem cells that survive oncotherapy is discussed. A novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs), developmentally equivalent to late migratory primordial germ cells, exists in adult gonads and survives oncotherapy due to their quiescent nature. However, the stem-cell niche gets compromised by oncotherapy. Transplanting niche cells (Sertoli or mesenchymal cells) can regenerate the non-functional gonads. This approach is safe, has resulted in the birth of fertile offspring in mice and could restore gonadal function early in life to support proper growth and later serve as a source of gametes. This newly emerging understanding on stem cells biology can obviate the need to bank gonadal tissue and fertility may also be restored in existing cancer survivors who were earlier deprived of gonadal tissue banking before oncotherapy.

Medical Rejuvenation in Georgia in the past: the Sukhumi Station.

If youth and body appearance enhancement is as old as Homo Sapiens, reliable medical technology for such activities is only about 100 years old. At the dawn of the 20th century, surgical operations performed under the Voronoff's treatment plan (monkey gonads' tissue grafting into humans) or the Steinach's technique (vasoligation) offered a promise of longevity, beauty and therefore youth restoration. The many links with a newly recognized discipline, endocrinology, offer a critical insight on the strong interactions between medicine and surgery in the promise of successful antiaging. On the front-line of scientific research, the Institute of Experimental Endocrinology's primate station in Sukhumi (West Georgia, now Abkhazia, on the Black Sea coast) developed a leadership role in the medical research, including rejuvenation with testis' tissues. Authors focus their attention to the everlasting commitment to experimental and clinical research as developed by Sukhumi scholars and the related moral, practical and ideological implications.

The fused toes locus is essential for somatic-germ cell interactions that foster germ cell maturation in developing gonads in mice.

Ovarian development absolutely depends on communication between somatic and germ cell components. In contrast, it is not until after birth that interactions between somatic and germ cells play an important role in testicular maturation and spermatogenesis. Previously, we discovered that Irx3 expression was localized specifically to female gonads during embryonic development; therefore, we sought to determine the function of this genetic locus in developing gonads of both sexes. The fused toes (Ft) mutant mouse is missing 1.6 Mb of chromosome 8, which includes the entire IrxB cluster (Irx3, Irx5, Irx6), Ftm, Fts, and Fto genes. Homozygote Ft mutant embryos die around embryonic day 13.5 (E13.5); therefore, to assess later development, we harvested gonads at E11.5 and transplanted them into nude mouse hosts. Our results show defects in somatic and germ cell maturation in developing gonads of both sexes. Testis development was normal initially; however, by 3-wk posttransplantation, expression of Sertoli and peritubular myoid cell markers were decreased. In many cases, gonocytes failed to migrate to structurally impaired basement membranes of seminiferous cords. Developmental abnormalities of the ovary appeared earlier and were more severe. Over time, the Ft mutant ovary formed very few primordial or primary follicles, which contained oocytes that failed to grow and were surrounded by scarce granulosa cells that expressed low levels of FOXL2. By 3 wk after transplantation, it was difficult to identify ovarian tissue in Ft mutant ovary transplants. In summary, we conclude that the Ft locus contains genes essential for somatic-germ cell interactions, without which the germ cell niche fails to mature in both sexes.

Gonadal tissue cryopreservation and transplantation.

Transplantation of ovarian and testicular tissue has been practised for over a century, mainly for experimental purposes. It is now being considered as a potential strategy for preserving fertility in young patients, including children, undergoing sterilizing treatment for cancer and other diseases. Ovarian tissue biopsies can be stored at liquid nitrogen temperatures indefinitely so that, after thawing, they can be returned as either ortho- or heterotopic grafts to the original patient. A different approach is needed for preserving male germ cells to restore fertile potential. Experimental studies have shown that spermatogonial stem cells injected into the rete testis/seminiferous tubules can re-initiate spermatogenesis after sterilizing treatment with alkylating agents; alternatively, in prepubertal cases, testicular biopsies that have been cryopreserved can be grafted subcutaneously to generate enough spermatozoa for intracytoplasmic sperm injection (ICSI). These strategies have been demonstrated in animal models and are now undergoing clinical testing.

Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.

The AML1:CBFbeta transcription factor complex is essential for definitive hematopoiesis. Null mutations in mouse AML1 result in midgestational lethality with a complete lack of fetal liver hematopoiesis. While the cell autonomous nature and expression pattern of AML1 suggest an intrinsic role for this transcription factor in the developing hematopoietic system, no direct link to a functional cell type has been made. Here, we examine the consequences of AML1 loss in hematopoietic stem cells (HSC) of the mouse embryo. We demonstrate an absolute requirement for AML1 in functional HSCs. Moreover, haploinsufficiency results in a dramatic change in the temporal and spatial distribution of HSCs, leading to their early appearance in the normal position in the aorta-gonad-mesonephros region and also in the yolk sac.

Amphibian sex determination and sex reversal.

Amphibians employ a genetic mechanism of sex determination, according to all available information on sex chromosomes or breeding tests. Sex reversal allows breeding tests to establish which sex is heterogametic and provides an indication of the mechanism of sex determination. Cases of spontaneous and experimental sex reversal (by temperature, hormones or surgery) are reviewed and illustrated by previously unpublished studies on crested newts. These newts respond conventionally to temperature and hormone treatment but provide anomalous results from breeding tests. It is suggested that both the evolution from temperature dependency to a genetic switch and from ZZ/ZW to XX/XY are superimposed on a generally uniform mechanism of sex determination in all vertebrates.

Artificial reproduction and the family of the future.

The techniques of artificial reproduction have until fairly recently been geared to overcoming male sterility by means of AID and IPSI and female sterility by way of GIFT, VISPER and DIPI. The above techniques have concentrated on achieving conception either within or without the uterus. Gestation has always been completed in utero and the therapy was reserved for married couples. The above circumstances only required limited legal regulation and its effect on the family unit was minimal because the child was mostly conceived from the gametes of its parents New technologies which are looming on the horizon, however, threaten to change the concept of parent and family radically. I refer to the imminent perfection of the artificial uterus and the cloning of human cells. If these technologies are sanctioned, a child would be conceived from the cells of one parent only and will not be gestated within the mother's womb. How will society and the law react to these technologies? Will they regulate them or proscribe them? I will argue in favour of the former rather than the latter.

Schistosoma mansoni infection inhibits maturation of ovotestis allografts in Biomphalaria glabrata (Mollusca:Pulmonata).

The posterior tip of juvenile snails, consisting of immature ovotestis, vesicular connective tissue, and digestive gland acini, all surrounded by body wall, was implanted into the hemocoel of allogeneic adult Biomphalaria glabrata. Recipient snails were either uninfected or had been infected 15 days previously with Schistosoma mansoni. Graft recipients were dissected at 30 or 60 days postimplantation (DPI), and the implants, along with preimplantation controls, were examined histologically. The gonads in preimplantation controls consisted of a partially coiled collecting canal partitioned dorsally by connective tissue septa and contained mainly spermatogonia-like cells and small oocytes. In uninfected recipients, implanted gonads underwent rapid development, i.e., had formed mature acini and were carrying out vigorous gametogenesis, both spermatogenesis and oogenesis, by 30 DPI. At 60 DPI, numbers of spermatozoa had increased substantially in most implants. In infected recipients, relatively little development or gametogenesis occurred at either 30 or 60 DPI, and oogenesis was more strongly inhibited than spermatogenesis. Except for 1 implanted gonad (out of 25) that had undergone necrosis at 60 DPI and a second otherwise healthy 60-day implant partially encapsulated at its cut surface (both in uninfected recipients), no significant hemocytic response occurred against the allografts.

Insights into pigmentary phenomena provided by grafting and chimera formation in the axolotl.

The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.

Fetal allograft survival in immunocompetent recipients is age dependent and organ specific.

This study explores whether fetal allograft survival is age dependent and organ specific. Fetal rat tissue (renal, gonadal, hepatic) from the third trimester of gestation (days 15-21) was transplanted into 306 outbred adult rats for 10-30 days. Grafts were studied by morphometric and histologic analysis. Ten days after implantation, renal tissue (N = 75) from late gestation (days 19-21) showed no increase in size. In contrast, 17-day fetal grafts (N = 20) grew 6.8 +/- 3.4 times,* while 15-day fetal grafts (N = 28) grew 17.5 +/- 6.1* times. (The symbol "*" indicates p less than 0.05, compared to original size). Twenty days after implantation, these 15-day fetal grafts (N = 20) grew 48.8 +/- 17.7* times. Ten days after grafting, the younger fetal tissue showed excellent maturation of renal elements and no sign of rejection; older fetal grafts had poor renal architecture and a dense lymphocytic infiltrate. The 15-day fetal gonadal tissue (N = 18) showed a moderate 10.6 +/- 3.2* increase in size while the 15-day hepatic grafts (N = 16) were regularly rejected within 10 days. Selected fetal allografts from early in the third trimester can not only survive but can grow and mature in an immunocompetent recipient. This fetal graft growth appears to be both age dependent and organ specific. The use of fetal organs may broaden the potential pool for transplantation. However, further studies are needed to define the ontogeny of graft acceptance.

[Development of gonads transplanted homoplastically in limbs of sexually immature axolotls]. / Evolution des gonades transplantées homoplastiquement dans le membre d'Axolotl, sur des animaux sexuellement immatures

Graft development depends on sexual maturation of the host. Three successive steps in development of transplants have been demonstrated: somatic growth, gonial division and germinal maturation. Sexual maturity occurs earlier in females than in males.

[Evolution of xenografts of embryonic gonads and juvenile ovaries from Ambystoma mexicanum Shaw, transplanted into Triturus alpestris Laur]. / Evolution des xénogreffes de gonades embryonnaires et des ovaires juvéniles d'Ambystoma mexicanum Shaw, transplantés sur Triturus alpestris Laur

Gonad primordium of Ambystoma mexicanum when grafted at tail-bud stage on Triturus alpestris is indefinately tolerated. Testis or ovaries derived from those grafted embryonic gonads follow their differentiaion and reach sexual maturity. Axolotl juvenile ovaries transplanted in Triturus alpestris are also tolerated; the ovocytes achieve the entire vitellogenesis but are not able to be laid.

Experimental studies on a lethal gene (1) in the Mexican axolotl, Ambystoma mexicanum.

1. Gene l is a recessive lethal factor found in the white strain of axolotls. Animals heterozygous for the gene are phenotypically normal. When mated with each other they give offspring 25% of which exhibit the lethal effects of the gene. 2. The l/l homozygotes develop normally to an advanced embryonic stage (Harrison stage 40) before the effects of the gene are first manifested. They then come to display a characteristic combination of abnormalities, including a disproportionately small head, small and poorly developed eyes, abnormal poorly developed gills, undifferentiated limb buds, and reduced overall growth rate. They may feed briefly, but soon stop and invariably die within a few weeks of the time of hatching. 3. The action of gene l has been analyzed by parabiosing mutant and normal embryos, and by grafting various organ primordia reciprocally between mutant and normal embryos. Parabiosis to normal embryos fails to correct the abnormalities of the mutants, although their survival may be somewhat prolonged. Grafts of mutant organ primordia (eye, limb, gill, pronephros, gonad, head) also invariably fail to show improved development or to survive on normal hosts; normal organ primordia develop normally on mutant hosts so long as the mutant survives. These experiments indicate that gene l is a recessive autonomous cell lethal affecting all of the organ systems during late embryonic and early larval development.