The MRL mouse heart healing response shows donor dominance in allogeneic fetal liver chimeric mice

We previously demonstrated that after a severe cryoinjury to the right ventricle of the heart, adult MRL mice display structural and functional recovery with myocardial tissue replacement resembling that seen in amphibians. The control non-regenerating adult C57BL/6 (B6) mouse shows a predominant scar response. In the present study, radiation chimeras reconstituted with fetal liver cells from either healer MRL or nonhealer B6 mice were generated to test for a transfer of phenotype. Allogeneic MRL fetal liver cells were injected into x-irradiated (9 Gy) B6 mice and B6 fetal liver cells were injected into x-irradiated MRL mice. In these allogeneic chimeras, the healing response to cardiac cryoinjury was predominantly of the donor phenotype. Thus, MRL fetal liver cells transferred the healing phenotype to the B6 nonhealer with the appearance of Y-chromosome positive, donor-derived cardiomyocytes in the injury site and MRL-like healing with little scar. Similarly, B6 fetal liver cells transferred the nonhealing phenotype to the MRL with little cardiomyocyte growth and an acellular B6-like scar. These results are in contrast to the ear hole closure response which was of the recipient phenotype. We conclude that, in the case of the heart, fetal liver-derived stem cells regulate regenerative healing.     

Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally

Animal cloning by nuclear transfer has been successful in several species and was expected to become an alternative reproductive technique. Among the problems associated with this cloning technique, however, are its low success rate and high mortality of cloned animals even if they develop to term. Nuclear transfer has thus come to be considered too difficult to apply as a reproductive technique. The transplantation of male germ cells or pieces of testicular tissue has enabled the induction of spermatogenesis from fetal or postnatal male mice. In the present study, we examined whether functional male gametes could be obtained by the transplantation of pieces of testicular tissue from cloned mice that died immediately after birth with typical aberrant phenotypes, such as large offspring syndrome. Donor testicular tissues were retrieved from cloned mice that died postnatally and were transplanted into the testes of recipient nude mice. Two to three months after transplantation, the grafted donor testicular tissue had grown in the host testis, and histological analysis showed that spermatogenesis occurred within the graft. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted donor tissue were able to support full-term development of progeny. These results clearly showed that functional spermatogenesis could be induced by transplanting testicular tissue from cloned mice that died postnatally into recipient mice. The strategy presented here will be applicable to cloned animals of other species, because the xenografting of testicular tissue into mice has been demonstrated previously to be possible.     

胎肝造血干细胞在扩散盒培养条件下移植特性的变化

经体内扩散盒培养6d 后的 LACA 小鼠胎肝细胞移植给照射的同系成年小鼠,造血干细胞在受体脾脏和骨髓中的有效植入率比正常胎肝细胞明显提高。但这种效果在同种异基因受体小鼠中则完全消失。实验结果表明,个体发育屏障和移植免疫屏障是决定同种胎肝移植能否成功的两个重要因素。胎肝细胞经体内或体外培养后可以模拟造血干细胞在体内的发育成熟,从而增强对成年造血微环境的适应性。用短期体内培养的方法,可以改变胎肝造血干细胞的某些生理特性,从而减弱个体发育屏障,但不能克服胎肝同种移植中的免疫性抗力。为了保证同种胎肝移植的成功,必须进一步同时克服两种屏障。     

Mouse cloning with nucleus donor cells of different age and type

We have tested different cell types as sources for nucleus donors to determine differences in cloning efficiency. When donor nuclei were isolated from cumulus cells and injected into recipient oocytes from adult hybrid mice (B6D2F1 and B6C3F1), the success rate of cloning was 1.5-1.9%. When cumulus cell donor nuclei were isolated from adult inbred mice (C57BL/6, C3H/He, DBA/2, 129/SvJ, and 129/SvEvTac), reconstructed oocytes did not develop to full term or resulted in a very low success rate (0-0.3%) with the exception of 129 strains which yielded 0.7-1.4% live young. When fetal (13.5-15.5 dpc), ovarian, and testicular cells were used as nucleus donors, 2.2 and 1.0% of reconstructed oocytes developed into live offspring, respectively. When various types of adult somatic cells (fibroblasts, thymocytes, spleen cells, and macrophages) were used, oocytes receiving thymocyte nuclei never developed beyond implantation, whereas those receiving the nuclei of other cell types did. These results indicate that adult somatic cells are not necessarily inferior to younger cells (fetal and ES cells) in the context of mouse cloning. Although fetal cells are believed to have less genetic damage than adult somatic cells, the success rate of cloning using any cell types were very low. This may largely be due to technical problems and/or problems of genomic reprogramming by oocytes rather than the accumulation of mutational damage in adult somatic cells.     

Limiting dilution analysis of the stem cells for T cell lineage

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.     

Presence of mast cell precursors in fetal liver of mice

When liver cells obtained from 13- to 18-day embryos of beige (Chediak-Higashi syndrome) mice were transplanted into irradiated normal adult mice, tissue mast cells with giant granules showing beige mouse origin developed in the normal recipient mice. Mast cell precursors seem to have developed earlier in the liver of embryos than mast cells themselves since no mast cells were detectable in any tissues of 13- and 14-day embryos. This result suggests that tissue mast cells originate from hematopoietic tissues not only in adult mice but also in mouse embryos.     

Effect of syngenic lymphocytes on allogenic inhibition of hematopoietic stem cells of embryonic liver]

The transplantation of liver from the embryos and newborn C57BL-6 mice to the lethally irradiated hybrids (CBA X C57BL/6) F1resulted in 90% allogenic inhibition of the colony-forming activity of the donor elements. The degree of allogenic inhibition of liver cells of 19 days old embryos and newborn mice may be changed with the help of syngenic lymphocytes of adult mice or delayed transplantation of cells 72 hrs following the irradiation of recipients but these procedures proved to be ineffective with the liver cells of 13 and 16 days old embryos. A suggestion is put forward to the effect that the allogenic inhibition is based on the active reaction of recipient hybrids (CBAXXC57BL/6) F1 to the stem hemopoietic cells of C57BL/6 mice.     

Differentiation and grafting of haemopoietic stem cells from early postimplantation mouse embryos

Haemopoietic stem cells evidently arise in early post-implantation mouse embryos at day 6 of gestation, a day earlier than previously thought (Moore & Metcalf, 1970). Disaggregated embryonic cells were injected into mice given a lethal dose of X-irradiation. The presence of donor haemoglobin (Whitney, 1978) and donor lymphocytic glucose phosphate isomerase (GPI) (Siciliano & Shaw, 1976) to detect donor erythrocytes and lymphocytes, respectively, were monitored by starch gel electrophoresis. The presence of donor cells was also assessed by using donor embryos carrying the T6 marker chromosomes. Decidual cells dissected free of embryos did not colonize any recipients. Disaggregated cells from early mouse embryos first colonized the liver and then repopulated the haemopoietic systems of recipients, producing adult donor haemoglobin within 2-3 days and donor GPI within 3-5 days. 80% of grafted X-irradiated recipients survived and donor markers were found in each of them. All nongrafted controls died within 14 days of X-irradiation and none of them showed donor markers. Disaggregated embryonic cells could be grafted across major histocompatibility barriers unlike adult bone marrow. Haemopoietic stem cells could not be identified in disaggregated cells from embryos aged less than 6 days gestation.     

Effect of transplantation of thymus, bone marrow and spleen cells on the regenerative processes in pathologically changed liver]

In tests on CBAXC57BL mice with experimental hepatitis, induced by carbon tetrachloride, the transplantation of cells from the lymphoid organs of healthy donors intensified the repairing process in the recipient pathologically changed liver. The most pronounced normalization was seen in the liver of the animals given thymocytes which suggests a deficit of these cells in experimental toxic hepatitis and indicates a definite role of the thymus in the repairing processes of the damaged liver.     

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.     

Fetomaternal trafficking in the mouse increases as delivery approaches and is highest in the maternal lung

The purpose of the study was to understand in more detail the natural history of fetomaternal cell trafficking in healthy pregnant mice. Our goal was to identify the best target organs and days during pregnancy for further mechanistic studies of the role of fetal cells in maternal disease and injury. C57BL/6J wild-type virgin females (n = 54) were mated with congenic enhanced green fluorescent protein (EGFP) transgenic males. During pregnancy and after delivery, female mice were euthanized, and eight organs and blood were analyzed for the presence of fetal GFP+ cells with flow cytometry and real-time quantitative PCR. Maternal lungs, liver, and spleen were also analyzed by fluorescent stereomicroscopy. Fetal GFP+ cells were first found at low frequency at Embryonic Day 11, increased to a maximum at Embryonic Day 19, and decreased rapidly postpartum. These fetal cell dynamics were significantly reproducible among all mice studied. In addition, there was a consistent distribution of fetal cells within maternal organs, with lung, liver, blood, and spleen having the greatest concentrations; these were highly correlated at all time points (P < 0.0001). Maternal lung contained 10- to 100-fold more fetal cells than any other organ, and using all three techniques, the number of fetal cells detected was the most consistent and reproducible in this organ. Stereomicroscopy showed that within the lung, fetal cells were widely and apparently randomly distributed. Using a murine model, our data demonstrate that fetomaternal cellular trafficking occurs in reproducible patterns, is maximal near term delivery, and has predilection for the maternal lung.     

Th2 polarization in target organs is involved in the alleviation of pathological damage mediated by transplanting granulocyte colony-stimulating factor-primed donor T cells

Acute graft-versus-host disease(a GVHD) is caused by allo-activated donor T cells infiltrating target organs. As a regulator of immune function, granulocyte colony-stimulating factor(G-CSF) has been demonstrated to relieve the a GVHD reaction.However, the role of G-CSF-primed donor Tcells in specific target organs is still unknown. In this study, we employed a classical MHC-mismatched transplantation mouse model(C57BL/6 into BALB/c) and found that recipient mice transplanted with GCSF-primed T cells exhibited prolonged survival compared with that of the PBS-treated group. This protective function against GVHD mediated by G-CSF-primed donor T cells was further confirmed by decreased clinical and pathological scores in this a GVHD mouse model, especially in the lung and gut. Moreover, we found that Tcells polarized towards Th2 cells and regulatory T cells were increased in specific target organs. In addition, G-CSF treatment inhibited inducible co-stimulator(ICOS) expression and increased the expression of tolerance-related genes in recipient mice. Our study provides new insight into the immune regulatory effects of G-CSF on T cell-mediated a GVHD, especially for its precise regulation in GVHD target organs.     

Participation of transfused bone marrow cells in reparative osteohistogenesis

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.     

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.     

Transplanted neuronal precursors migrate and differentiate in the devel-oping mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of prolife     

Reversibility of hematopoietic stem cell direction (not 'commitment') as influenced by the microenvironment.

The 7-day colony types (E vs. G) formed in irradiated recipient spleens and bones by donor cells from adult bone marrow and spleen and early fetal liver were examined. Both direct and sequential transplant (retransplantation shortly after lodgment) experiments were carried out. It was found that recipient spleen receiving donor bone marrow, spleen or fetal liver developed significantly higher E/G ratios in that order, but that the E/G for colonies in recipient bones remained around 1. This led to the following conclusions concerning differences in the proportion of E or G colonies formed in recipient spleens and bones: (1) selective lodgment of 'committed' CFU-S does not occur; (2) selected repression or stimulation of 'committed' CFU-S does not occur; and (3) the findings are best explained by a condition of reversible directedness present in many or all transplantable pluripotent stem cells.     

Transplanted neuronal precursors migrate and differentiate in the developing mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of proliferation, which is likely to be induced by some environmental factors.     

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-γ in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-γ followed by administration of mouse IFN-γ (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.     

R‐spondin 1 and noggin facilitate expansion of resident stem cells from non‐damaged gallbladders

Pioneering studies within the last few years have allowed the in vitro expansion of tissue‐specific adult stem cells from a variety of endoderm‐derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R‐spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5‐positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non‐regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R‐spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder‐derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride‐treated mice. Human gallbladders were also a source of organoid‐forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.