骨髓间充质干细胞移植后在胸腺内的定居

BACKGROUND: Bone marrow mesenchymal stem cells have low immunogenicity and can induce immune tolerance. At present, the mechanism of immune regulation of bone marrow mesenchymal stem cells is not completely understood. It has been rarely reported whether the bone marrow mesenchymal stem cells can migrate to the thymus after transplantation.OBJECTIVE: To observe the distribution and survival of bone marrow mesenchymal stem cells in the thymus of aging rats after transplantation.METHODS:Bone marrow mesenchymal stem cells cultured in vitro were transfected by adenovirus vectors expressing green fluorescent protein. Transfected bone marrow mesenchymal stem cells were injected into the portal vein of aging rats. At days 3, 7, 14, 21 after transplantation, the survival of bone marrow mesenchymal stem cells homing to the thymus was observed under fluorescence microscope. At day 3 after transplantation, thymus tissues were taken and stained with hematoxylin-eosin for pathological observation. RESULTS AND CONCLUSION:Green fluorescent protein-labeled bone marrow mesenchymal stem cells had a strong green fluorescence at days 3 and 7 after transplantation, and the cell contour was clear. There was no significant difference in the mean absorbance values at days 3 and 7 (P > 0.05). Expression of green fluorescent protein was weakened significantly at days 14 and 21 compared with that at day 3 (P < 0.05). At 3 days after transplantation, the transplanted bone marrow mesenchymal stem cells were clearly visible in the thymus, and acute rejection was not observed. The results show that bone marrow mesenchymal stem cells can migrate to the damaged thymus tissue through the blood circulation, and can survive at least 1 week.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Conditioning of Nude bone marrow increases in vitro migration to thymus supernatant.

The thymus must be continuously seeded by cells termed prothymocytes in order to maintain normal T-cell development (Scollay et al. 1986). Using parabiotic studies, the source of prothymocytes appears to be either from the fetal liver or the adult bone marrow (Roderwald et al. 1992). The ability of the body to distinguish self from non-self and mount a functionally mature immune response is dependent upon the intrathymic education of these cells. Therefore, it is apparent that successful migration of prothymocytes into the thymus is an inescapable event in the development and maintenance of the immune system. Utilization of the athymic Nude mouse is a valuable asset in the elucidation of the mechanisms influencing the migration of bone marrow cells into the thymus. Its aberration enables investigators to examine the effect of thymic factors on cells previously devoid of thymic influence. In an attempt to understand the migration of normal prothymocytes into the thymus, we analyzed the in vitro migration of athymic bone marrow cells towards newborn thymus supernatant. Adult athymic murine bone marrow cells were incubated in either thymus supernatant or media and allowed to migrate toward one or the other. Similar control experiments were performed using CBA adult mice. Results indicate that athymic bone marrow migration towards both supernatant and media can be restored to control levels after incubation in thymus supernatant.     

Effect of age on the proportion of mouse bone marrow cells migrating in response to newborn thymus supernatant. Cell migration and thymus evolution in mice.

The effect of mouse age on the in vitro migration of hemopoietic precursor cells from bone marrow of C57BL/6 to thymic supernatant from newborn mice was studied to determine whether a reduction in the migratory readiness or number of precursor cells might at lest partly explain thymic involution due to aging. The percentage of bone marrow cells migrating to the thymic supernatants increased to age of 7 weeks, and then decreased progressively. Thymus weight underwent almost exactly the same evolution. The decline of the population of migration-ready T-precursor in adult mice may this explain age-related thymic involution.     

The entry of the prothymocyte into the thymus after lethal irradiation and bone marrow transplantation. I. Seeding of bone marrow cells into the thymus

Bone marrow (BM) cells arrive in the thymus of lethally irradiated mice as early as three hours after bone marrow transplantation (BMT). They can be recognized by labeling of the injected cells with Hoechst 33342 (direct homing assay). In order to relate the immigrated BM cells to thymocyte precursor cells, direct homing and thymus repopulation experiments were compared. It was shown that homing of BM cells depends on the time between lethal irradiation and BMT, while it was previously shown that thymus repopulation does not. In addition, thymic immigrants were smaller than precursor cells committed to the T cell limeage (prothymocytes) and their progenitors. A cell population obtained from normal BM cells and enriched in stem cells (purified stem cells) was previously shown to repopulate the thymus similarly as BM cells from mice pretreated in vivo with 5-fluorouracil (FUBM). Both cell suspension showed a delayed thymus repopulation when compared to normal BM. This is indicative for a depletion of prothymocytes in these cell suspensions. In the direct homing assay, however, it was found that relatively many cells from FUBM seeded into the thymus, while purified stem cells did not. These results indicate that most if not all donor cells that are present in the thymus at three hours after BMT are not thymocyte precursor cells.     

In vivo homing of thymus-enriched bone marrow cells

A population of adult CBA/J mouse bone marrow (BM) cells enriched by in vitro migration to supernatant prepared from neonatal thymus was labeled with a DNA-binding fluorochrome, Hoechst dye No. 33342 (H33342). Labeled cells were injected into irradiated recipients in order to compare the in vivo localization of the migration-enriched BM (MEBM) cells to the localization of injected nonenriched BM (NEBM) cell controls. A characteristic difference in the distribution of localized cells was observed in the spleen but not in other lymphoid organs. At 2 hr after injection the MEBM cells were located in the marginal zones surrounding the periarterial lymphoid sheaths (PALS) of the splenic white pulp. At 6 hr after injection the MEBM cells were seen distributed between marginal zones and the PALS and by 16 hr they had localized almost exclusively in the white pulp. In contrast, the NEBM cells were located in the marginal zones or red pulp for the duration of the experiment. These observations show that the MEBM cells home selectively to T-cell areas of the spleen. Direct immunofluorescent monoclonal antibody staining of H33342-labeled cells obtained from the recipient spleens at 16 hr demonstrated that the MEBM cells were negative for Thy-1 antigen, indicating that acquisition of Thy-1 was not prerequisite to the observed homing. The results are compared to known localization patterns of mature lymphocytes.     

Adhesion and homing of blood-borne cells in bone marrow microvessels.

After birth, the bone marrow (BM) is the principal site of hematopoiesis in mammals. Thus, a large number of newly formed blood cells must penetrate the wall of BM microvessels to enter the circulation. In addition, the BM appears to function as a lymphoid organ and is also part of the macrophagal system. Subsets of circulating lymphocytes and other cells of the immune system continuously home to the BM. However, neither the mechanisms of blood cell migration to and from the BM nor its precise role in the immune system are well understood. One reason for the relative paucity of data on BM physiology is the fact that normal BM is surrounded by thick cortical bone that impedes direct observation and experimental manipulation. One notable exception is the calvaria of the murine skull where hematopoietically active BM is only covered by a thin lamella of bone that is sufficiently translucent to allow a detailed in situ analysis of the BM microcirculation by epi-fluorescence microscopy. Here, we review our current knowledge of the anatomic, hemodynamic, and endothelial properties of the specialized microvascular bed within murine skull BM. In addition, we summarize recent studies on the molecular mechanisms that mediate the homing of circulating hematopoietic progenitor cells to the BM, an event that is critical for the success of BM transplantations.     

The effects of several diphosphonates on murine bone marrow proliferation

Six diphosphonates were examined for their ability to alter proliferative responses of mouse bone marrow cells to recombinant human M-CSF and recombinant murine GM-CSF. Risedronate ([2-(3-pyridinyl)-ethylidene] hydroxy bisphosphonic acid) added toin vitro cultures at 10 μM, suppressed the response to M-CSF by 58%, but had no significant effect on GM-CSF-induced proliferation. Ethane-1-hydroxy-1,1-bisphosphonic acid (EHDP), dichloromethylene bisphosphonic acid (Cl₂MBP), 3-amino-1-hydroxy-propylidene-1,1-bisphosphonic acid (APD), (4-chlorophenyl)-thiomethylene bisphosphonic acid (tiludronate) and (cycloheptylamino)-methylene bisphosphonic acid (YM-175) had no significant effect. Treatment of mice for 5 or 14 days with 200 mg/kg/day p.o., Cl₂MBP or 3 mg/kg/day p.o. of risedronate failed to inhibit M-CSF- or GM-CSF-induced proliferation by recovered bone marrow cells. Addition of Cl₂MBP or risedronatein vitro to these cells did not reveal any change in sensitivity to CSFs as a result of exposure to diphosphonatein vivo.

     

The role of hematogenous and intrinsic precursor cells in lymphocyte production in murine bone marrow and thymus

It is well recognized that the bone marrow contains cells that can repopulate a depleted thymus as well as cells that can be induced to express phenotypic markers characteristic of T cells. It is not known, however, to what extent thymocytopoiesis in the normal thymus relies on immigrant, bone marrow-derived cells, nor whether some T cell precursors have entered the bone marrow from the circulation. We used the parabiotic system to test whether thymocytopoiesis relies on progenitors intrinsic to the thymus or on cells that enter the organ from the circulation. In the same system, we have also investigated whether Thy-1^? bone marrow lymphocytes that respond to phytohemagglutinin (PHA) by proliferation and Thy-1 expression are produced by my-elogenous or hematogenous progenitors. Syngeneic CBA/HT6 and CBA/CaJ mice were joined in parabiotic union at 4–6 weeks of age. Cross circulation between the two partners was verified by the equilibration of Evans' blue dye injected into one partner and by the equilibration of PHA-responsive T cells in the spleen of the parabionts. Chromosome spreads were prepared from the PHA-stimulated T cell-depleted bone marrow and from spontaneously proliferating thymocytes as well as from thymocytes stimulated by PHA or Concanavalin A (Con A). The exchange of spleen colony-forming units (CFU-S) in the femoral marrow was assessed by karyotyping individual spleen colonies. Regardless of the length of parabiotic union, ranging from 4 to 20 weeks, Thy-1^?, PHA-responsive bone marrow lymphocytes remained predominantly of the host type with only 3% being derived from the opposite partner. The same held true for CFU-S in the femur; only around 5% of this cell population were of the nonhost type. Thus, although some Thy-1^?, PHA-responsive lymphocytes in the bone marrow may be derived from hematogenous stem cells, the majority of them are generated by precursors resident in the bone marrow. Likewise, regardless of the length of parabiotic union, at least 95% of spontaneously proliferating cells in the thymus of each partner possessed the karyotype of the host, and this held true also for PHA- or Con A-stimulated thymocytes, indicating that the small population of spontaneously proliferating immigrant cells cannot account for the production of the large number of postmitotic (mitogen-responsive) thymocytes. Our findings, therefore, demonstrate a high degree of self-maintenance for Thy-1^?, PHA-reponsive lymphocytes in the bone marrow and also for intrathymic T cell precursors. In the unperturbed, postnatal thymus, thymoctye production does not rely on cell input from the circulation; the vast majority of thymoctyes are generated by an intrathymic precursor pool that is independent of immigrant myelogenous T cell precursors.     

Effect of In vivo administration of cisplatin on the colony forming ability of murine bone marrow cells

In vitro colony forming ability of bone marrow cells obtained from cisplatin-treated C3H/He mice was studied. Mice were administered cisplatin in a single intraperitoneal dose of 10 mg/kg body wt, 24 h prior to the harvest of femoral bone marrow cells. Incubation of untreated bone marrow cells without any CSF in vitro showed little colony forming ability which was marginally enhanced in cisplatin-treated bone marrow cells. Presence of M-CSF (250 U/ml) or GM-CSF (250 U/ml) in the culture medium significantly augmented the colony forming ability of both untreated and cisplatin-treated bone marrow cells. In the presence of M-CSF, colony forming units - macrophage (CFU-M) were predominantly high in untreated bone marrow cells, followed with CFU - granulocyte - macrophage (CFU-GM). The number of CFU-M was significantly up-regulated in response to M-CSF in bone marrow cells obtained from cisplatin administered mice, whereas the number of CFU-GM remained unchanged, as compared to untreated mice. Both CFU-M and CFU-GM were enhanced in the presence of GM-CSF in untreated bone marrow cells. Cisplatin-treated bone marrow cells on incubation in the presence of GM-CSF showed a significant enhancement of CFU-M and GM as compared to untreated samples. IL-1 (100 U/ml) in the presence of M-CSF significantly up-regulated colony forming ability of cisplatin-treated bone marrow cells, whereas TNF (100 U/ml) inhibited the colony forming ability. These effects of IL-1 and TNF on the in vitro colony formation of cisplatin-treated bone marrow cells were reversed in the presence of anti-IL-1 and anti-TNF antibodies, respectively.     

系统性红斑狼疮患者骨髓细胞培养上清对间充质干细胞衰老作用的研究

为了探讨SLE患者骨髓局部微环境对间充质干细胞(MSC)衰老的影响及机制。首先收集性别、年龄匹配的健康对照者及SLE患者骨髓各6例,留取上清液并灭活补体;以含10%骨髓上清的DMEM/F12培养基模拟机体骨髓局部微环境,培养脐带来源的MSC;SA-β-gal染色并计数阳性MSC比例;real time PCR及western blot法分别检测MSC的p53、p21基因及蛋白表达水平;荧光显微镜观察MSC核内DNA损伤标记53BP1的表达情况;CCK8检测细胞活力;流式细胞术检测Annexin V/PI评估细胞凋亡。结果显示,与正常对照相比,SLE患者骨髓上清可显著增加β半乳糖苷酶阳性MSC细胞比例,且上调p53和p21的表达量;在SLE患者骨髓上清处理的条件下,MSC细胞内与DNA损伤反应有关的标志性蛋白53BP1的表达水平显著增高;SLE患者骨髓上清还能够明显抑制MSC的增殖能力。这提示SLE患者骨髓上清促进MSC衰老,其机制可能在于诱导MSC细胞核内DNA损伤反应。     

MHC restriction of murine T lymphocyte reactivity analysed by growth of bone marrow cells in vitro on thymus epithelial monolayers.

Mature mouse T lymphocytes, derived from long-term culture of bone marrow cells on thymus epithelial monolayers, were analysed with respect to their ability to co-operate with B cells (for antibody production) or T cells (in the generation of cytotoxic cells) when the bone marrow T precursor cells and the thymus epithelial cells differ at defined regions of the major histocompatibility complex. A pool of more mature bone marrow T-cell precursors gave rise to T cells interacting only with T/B lymphocytes sharing MHC determinants with the strain of origin of the bone marrow cells used. In contrast, a more immature bone marrow T-cell precursor pool produced T lymphocytes which had acquired MHC restriction (in terms of co-operativity with T/B cells) as defined by the MHC determinants of the thymus epithelium, and not those MHC determinants of the cultured bone marrow population. In addition, some evidence was obtained for Ir gene control (mapping in the MHC region) in the development of the repertoire of T cells involved in production of cytotoxic responses in vitro to TNP-modified self antigens.     

The initial characterization of a thymus factor chemotactic to bone marrow cells

Thymus supernatants were produced by cluturing minced newborn CBA/J mouse thymuses in serum-free media for 48 h. Supernatants thus obtained were chemotactic to a subset of bone marrow cells as assessed in blind well chambers, and enriched for immature lymphoid cells in the migrating cell population. The enriched population of cells was shown to be capable of homing to the thymus of an irradiated mouse in vivo in a significantly higher percentage than nonmigrated bone marrow cells. In this report, initial characterization of the factor(s) responsible for this in vitro migration is presented. Several well studied thymic factors were compared with the thymus supernatants for their ability to induce migration of bone marrow cells in vitro. Thymulin (FTS-Zn), FTS, and TP-5 (the pentapeptide fragment of thymopoietin) were used. None of these factors demonstrated chemotactic properties in the migration assay using concentration ranges in which other in vitro activities have been observed. The chemoattractive activity of the supernatant was unaltered by ultracentrifugation. The effects of temperature on the chemotactic properties of thymus supernatant were examined, and a fifty percent decrease in observed migration occurred with thymus supernatant heated to 100 degrees C for 1 h. In addition, incubation of the supernatant for 1 h at 37 degrees C with chymotrypsin, but not with trypsin, inhibited migration, presumably by inactivation of the active factor. Using Amicon microconcentrators, the supernatant was separated into several fractions based on molecular weight. Initial data suggest that the active fraction is in the less than 10,000 mw range.     

The thymus in patients with allogeneic bone marrow transplants.

The thymus glands from 11 patients with aplastic anemia or acute leukemia who received allogeneic bone marrow transplants were studied at autopsy. All showed marked cortical involution. In the short-term survivors the medulla and perivascular spaces were lymphocyte-depleted and the epithelial cells formed pseudorosettes. In those surviving over 2 months, increasing numbers of small lymphocytes were present, presumably reconstituted with donor lymphocytes. Phagocytosis of cellular debris was frequent, especially in patients with graft-versus-host reaction (GVHR) or treated with anithymocyte globulin (ATG). Plasma cells were numerous in perilobular tissue and were occasionally found within the medulla. The findings are compatible with the concept that the thymus plays an important role in the immune deficiency experienced after allogeneic bone marrow transplantation and in the subsequent lymphoid reconstitution.     

Differentiation of lymphocytes in the mouse bone marrow: III. The adoptive response of bone marrow cells to a thymus cell-independent antigen*

The response of mouse spleen cells to the T cell-independent antigen dinitrophenylated polymer of flagellin (DNP—POL), has been studied using an adoptive transfer system, and compared with the response of bone marrow cells. Spleen cells showed a complex cell dose—response relationship, with a markedly discontinuous curve, for assays performed before day 9 after transfer and antigen challenge. This discontinuity could be explained by a delay in attainment of the peak response for lower cell inocula. The curve became linear on a log—log scale when spleens were harvested on days 9 and 10 post-transfer.

Bone marrow cells gave a lower response than would be expected from their lymphocyte content. This response increased progressively with a delay before antigen challenge in the irradiated recipient or in tissue culture prior to cell transfer, suggesting a functional maturation in this cell population, whereas the performance of spleen cells fell off under similar circumstances. The findings were consistent with, but could not prove, the hypothesis that the immediate precursors of anti-DNP antibody-forming cells in bone marrow were high surface immunoglobulin density small lymphocytes that had arisen locally from precursors lacking detectable surface immunoglobulin, by a non-mitotic maturation.

     

A novel subset of murine B cells that expresses unmasked forms of CD22 is enriched in the bone marrow: implications for B-cell homing to the bone marrow

CD22 is a B-cell-restricted transmembrane protein, which acts as a negative regulator of B-cell signalling. CD22 also has lectin-like adhesive properties. When expressed on transfected fibroblasts, it is capable of mediating adhesion to other cells via recognition of cell-surface glycoconjugates terminating in alpha2,6-linked sialic acids. In previous studies in the mouse, CD22 was implicated as a bone marrow homing receptor for recirculating immunoglobulin D+ (IgD+) B cells through recognition of sialylated ligands on marrow sinusoidal endothelium. As the adhesive function of CD22 can be masked when alpha2,6-linked sialic acids are co-expressed at the cell surface, the aim of the present study was to investigate whether recirculating B cells have unmasked forms of CD22 that could be involved in bone marrow homing. Using alpha2,6-sialyllactose coupled to biotinylated polyacrylamide as a probe for detection of unmasked CD22, we showed that approximately 2-5% of IgD+ murine B cells in the spleen and mesenteric lymph nodes were able to bind this synthetic ligand. In the bone marrow, however, the fraction of IgD+ B cells with unmasked CD22 was increased by two- to fivefold. B cells from CD22-deficient mice were not stained with the polyacrylamide probe, confirming that staining of B cells in wild-type mice was caused by CD22 and not by other potential sialic acid-binding lectins. In conclusion, we have identified a new subset of mature B cells in the mouse with unmasked CD22. This subset of recirculating B cells may bind to CD22 ligands on bone marrow sinusoidal endothelium, leading to their selective homing and subsequent enrichment in this tissue.     

Immunohistology of the thymus in bone marrow transplant recipients.

The immunohistological findings in the thymus after bone marrow transplantation were studied in autopsy samples from 12 patients who had received allogeneic grafts as treatment for acute leukemia. The findings were compared with those in samples from normal individuals and from conventionally treated leukemic patients. The thymuses were hyperinvoluted in all grafted and nongrafted subjects. The remnants were composed of subcapsular and medullary epithelium which expressed the same phenotype as the normal thymus controls. Most samples also contained small remnants of cortical epithelium which lacked normal expression of HLA-DR antigens. The intraepithelial and stromal thymic lymphocytes were virtually all mature T cells, and no immature cortical thymocytes were seen. With the use of HLA-typing methods in 2 recipients of one-haplotype-matched grafts no donor cells could be identified in any of the thymic components. These findings provide no evidence for a functional role for the thymus in the differentiation of donor-derived precursor T cells, at least in the early period after transplantation.     

Characterization studies of suppressor cells in murine bone marrow chimaeras.

When BALB/c bone marrow cells were transferred to lethally X-irradiated C3H/He mice either intrasplenically (i.s.) or intravenously (i.v.), suppressor cells were detected by means of MLR coculture assays in the spleen of i.s. and i.v. chimaeras. Some but not all of the suppressor cells expressed a Thy 1.2 antigen, indicating that suppressor cells either sensitive or insensitive to anti-Thy 1.2 antibody plus complement treatment were generated in the spleen of i.s. and i.v. chimaeras. According to the examination of Lyt alloantigen expression on suppressor T cells, Lyt 1+2-, Lyt 1-2+, and Lyt 1+2+ suppressor T cells appeared to be present. The culture supernatants from several T-cell clones showed the suppressor activity against alloreactive MLR. Cell surface markers of these clones were composed of Lyt 1+2-, Lyt 1+2+ and Lyt 1-2+. In addition, since Carrageenan treatment abrogated the suppressor activity of plastic dish adherent cells, we conclude that some of them were composed mainly of macrophages.