Donor Bone Marrow-Derived T Cells Inhibit GVHD Induced by Donor Lymphocyte Infusion in Established Mixed Allogeneic Hematopoietic Chimeras

Delayed administration of donor lymphocyte infusion (DLI) to established mixed chimeras has been shown to achieve anti-tumor responses without graft-vs.-host disease (GVHD). Herein we show that de novo donor BM-derived T cells that are tolerant of the recipients are important in preventing GVHD in mixed chimeras receiving delayed DLI. Mixed chimeras lacking donor BM-derived T cells developed significantly more severe GVHD than those with donor BM-derived T cells after DLI, even though both groups had comparable levels of total T cells at the time of DLI. Post-DLI depletion of donor BM-derived T cells in mixed chimeras, as late as 20 days after DLI, also provoked severe GVHD. Although both CD4 and CD8 T cells contributed to the protection, the latter were significantly more effective, suggesting that inhibition of GVHD was not mainly mediated by CD4 regulatory T cells. The lack of donor BM-derived T cells was associated with markedly increased accumulation of DLI-derived alloreactive T cells in parenchymal GVHD target tissues. Thus, donor BM-derived T cells are an important factor in determining the risk of GVHD and therefore, offer a potential therapeutic target for preventing and ameliorating GVHD in the setting of delayed DLI in established mixed chimeras.     

Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule L^d were transplanted into the L^d gene deletion mutant BALB/c-H-2^dm2, an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of L^d-positive donor-derived and L^d-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against L^d. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by L^d within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.Cytomegaloviruses (CMV) are kept under tight immune control (for reviews, see references 22 and 23). As a consequence, acute CMV infection is resolved rapidly and does not result in disease unless the host is immunologically immature or immunocompromised. Bone marrow (BM) transplantation (BMT) as a therapy of hematological malignancies is associated with a transient immunodeficiency. Accordingly, during the period of immunocompromise, transmission of donor-type CMV with the transplant as well as recurrence of CMV from latency established within the organs of the transplantation recipient both entail a risk for destructive virus replication in tissues resulting in multiple-organ CMV disease (16). In BMT recipients, CMV-induced interstitial pneumonia is a frequent and endangering manifestation of CMV disease (11, 27). However, CMV infection does not inevitably result in fatal disease. It appears that CD8 T-cell reconstitution is the decisive parameter in the control of CMV after BMT. Clinical data have shown that both efficient reconstitution of CD8 T cells (41) and supplementation of antiviral CD8 T cells by preemptive cytoimmunotherapy with T-cell lines (42, 50) correlate with a reduced risk of human CMV disease, whereas combined in vivo-ex vivo T-cell depletion, intended as a prophylaxis against graft-versus-host (GvH) disease, accidentally resulted in an increased incidence of CMV infections in BMT patients (14). Aspects of these clinical problems can be approached experimentally in a murine model of BMT and concurrent infection with murine CMV (for an overview, see reference 35). Specifically, depletion of CD8 T cells, but not of CD4 T cells, performed in vivo during the phase of reconstitution after BMT abolished the development of protective antiviral immunity, with an inevitably lethal outcome (34, 47) resulting from multiple-organ pathology (34), including BM aplasia (29, 30). Likewise, an insufficient endogenous reconstitution was successfully supplemented by experimental adoptive cytoimmunotherapy with antiviral CD8 T cells. Again, CD4 T cells were not effective (36, 37, 39, 47). Altogether, clinical data on human CMV infection and experimental data from the murine model have so far been concordant and have identified CD8 T cells as the principal effectors controlling CMV infections after BMT.These findings imply that all conditions which lower the efficacy of CD8 T-cell reconstitution will increase the risk for progression of asymptomatic CMV infection to fatal CMV disease. Histoincompatibility between graft and recipient is a factor likely to negatively influence the restoration of antiviral immunity. Accordingly, even though cases of severe human CMV disease have been reported also after autologous BMT (27, 40), the incidence of CMV-related complications is generally higher after histoincompatible BMT (51). In clinical BMT, donor and recipient are usually matched in major histocompatibility complex (MHC) class II molecules, whereas differences in minor histocompatibility loci and in MHC class I loci are tolerated if unavoidable. Complications caused in the CMV-infected recipient by histoincompatibility may include (i) an impaired engraftment of transplanted cells in the recipient BM stroma, (ii) an immunological GvH response as well as a host-versus-graft (HvG) response directed against the foreign minor or major histocompatibility molecules, and (iii) a lack of antiviral T-cell control resulting from an inappropriate repertoire of viral antigenic peptides presented by infected tissue cells of the transplantation recipient.In a first attempt to dissect these possibilities, we have established a murine model of experimental BMT performed across a single MHC class I disparity, namely, the presence and absence of the L^d molecule in BALB/c mice (MHC class I molecules K^d, D^d, and L^d) and the L^d gene deletion mutant BALB/c-H-2^dm2 (44), respectively. Depending on the choice of donor and recipient for the BMT, immunogenetical GvH and HvG conditions can be studied separately (35). Work presented herein focuses on the HvG setting with BALB/c as the donor strain and the mutant as the recipient. Hence, after incomplete depletion of hematopoietic cells of the recipients, this model entails a risk for graft rejection caused by a recipient response directed against the donor MHC class I molecule L^d. In addition, presentation of viral peptides by L^d, including the immunodominant IE1 nonapeptide of murine CMV (18, 38), is confined to donor-derived hematopoietic cells and their progeny, whereas the parenchymal and stromal sites of cytocidal infection (34) lack L^d as the prevailing peptide presenter. The aim of the study was to investigate the influence of this particular MHC class I disparity on the control of murine CMV after BMT.     

Cytotoxic CD8+ T Cells Stimulate Hematopoietic Progenitors by Promoting Cytokine Release from Bone Marrow Mesenchymal Stromal Cells

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Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes

Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×10⁵EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.     

Prostaglandin E2 Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E₂ (PGE₂) production was greater in BMT mice than in untransplanted controls, but BMT using PGE₂-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE₂ analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-β expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE₂ overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE₂-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.     

Thy.1lowCD3− Cells Sorted from Nylon Wool-Passed Bone Marrow Cells Can Augment the H-2 Identical but Not Non-Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures

Thy. 1^lowCD3^– cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-α/β and -γ/δ on their cell surfaces. An extremely limited number of B10.BR (H-2^k) responder lymph node (LN) cells were stimulated with B10. D2 (H-2^d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1^lowCD3^– NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2^b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1^lowCD3^– NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2⁺ cells or Thy.1⁺ L3T4^– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1^low CD3^– NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1⁺ Lyt-2^– helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.     

Bone Marrow Stromal Antigen 2 (BST-2) DNA Is Demethylated in Breast Tumors and Breast Cancer Cells

Background

Bone marrow stromal antigen 2 (BST-2) is a known anti-viral gene that has been recently identified to be overexpressed in many cancers, including breast cancer. BST-2 is critical for the invasiveness of breast cancer cells and the formation of metastasis in vivo. Although the regulation of BST-2 in immune cells is unraveling, it is unknown how BST-2 expression is regulated in breast cancer. We hypothesized that meta-analyses of BST-2 gene expression and BST-2 DNA methylation profiles would illuminate mechanisms regulating elevated BST-2 expression in breast tumor tissues and cells.

Materials and Methods

We performed comprehensive meta-analyses of BST-2 gene expression and BST-2 DNA methylation in The Cancer Genome Atlas (TCGA) and various Gene Expression Omnibus (GEO) datasets. BST-2 expression levels and BST-2 DNA methylation status at specific CpG sites on the BST-2 gene were compared for various breast tumor molecular subtypes and breast cancer cell lines.

Results

We show that BST-2 gene expression is inversely associated with the methylation status at specific CpG sites in primary breast cancer specimens and breast cancer cell lines. BST-2 demethylation is significantly more prevalent in primary tumors and cancer cells than in normal breast tissues or normal mammary epithelial cells. Demethylation of the BST-2 gene significantly correlates with its mRNA expression. These studies provide the initial evidence that significant differences exist in BST-2 DNA methylation patterns between breast tumors and normal breast tissues, and that BST-2 expression patterns in tumors and cancer cells correlate with hypomethylated BST-2 DNA.

Conclusion

Our study suggests that the DNA methylation pattern and expression of BST-2 may play a role in disease pathogenesis and could serve as a biomarker for the diagnosis of breast cancer.     

Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

Background

Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells.

Methods

MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed.

Results

The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs.

Conclusion

PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs.     

Alkylating Damage by Dipin of Hematopoietic and Stromal Cells of the Bone Marrow

Effect of alkylating agent dipin was studied on hematopoietic (CFU-S) and stromal (CFU-F) progenitor cells. Single administration of dipin (0.06 mg/g) to adult (CBA × C57Bl/6) F1 hybrid mice induced a long-term (2 years) oscillations in the numbers of day 7 CFU-S and day 11 CFU-S in the bone marrow and spleen. Dipin also damaged the hematopoietic stroma as indicated by decreased numbers of CFU-F which remained low for at least a year. The capacity of stromal cells to form ectopic hematopoietic foci was considerably decreased and also remained low for 10 months. The obtained data suggest high dipin sensitivity of the earliest hematopoietic and stromal cells. The dynamics of CFU-S numbers in the hematopoietic organs supports their functioning on the basis of clonal succession (Kay, 1965).__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 267–272.Original Russian Text Copyright © 2005 by Domaratskaya, Bueverova, Payushina, Starostin.     

人骨髓基质细胞体外分离及定向培养内皮细胞

用Ficoll(比重1.077 g/ml)从正常成人骨髓中分离骨髓基质细胞(BMSCs),DMEM-HG 培养基内含20?S、GM-CSF(100 u/ml)、VEGF(10 ng/ml)、FGF(5 ng/ml)、L-谷氨酰胺(2mmol/ L)、肝素(90 u/ml),以及抗生素液进行定向培养和扩增其中的内皮细胞(ECs),Ⅷ因子相关抗原的免疫组化法和透射电镜观察(TEM)鉴定其细胞的性质。结果5.0×105个BMSCs在体外经定向ECs 培养和扩增8代后,获得了6.0×109个ECs,扩增了约1.2×104倍。70%-80%的细胞对Ⅷ因子相关抗原免疫组化呈阳性反应;光镜下细胞呈典型的“鹅卵石”样;TEM下可观察到胞浆内有Weible- palade小体,证实为内皮细胞。实验表明,BMSCs在体外分离和定向培养的ECs,经扩增后可能是心血管组织工程所需种子细胞的又一个重要来源。     

Similarity of the carbohydrate structures of H-2 and Ia glycoproteins

The glycopeptides produced by pronase digestion of two H-2K, two H-2D, and three Ia glycoprotein antigens were examined for size and charge. The glycopeptides derived from all of the antigens examined were found to have m.w. of 3250 +/- 200 daltons with a similar and variable composition of sialic acid residues. These data, when combined with the similarity in monosaccharide incorporation, suggest that the general parameters of the carbohydrate structure of the Ia glycoproteins from different I subregions and H-2 glycoproteins are highly similar if not identical.     

Isolation and analysis of H-2 and Ia alloantigens from wild mouse strains.

Immunoprecipitation and SDS-PAGE analysis were used to examine H-2 and Ia antigens from mouse strains with wild-derived MHC haplotypes. Antisera raised against the wild-derived strains contained anti-H-2 and anti-Ia antibodies which precipitated antigen molecules readily distinguishable by a single assay procedure. The antibodies to wild strains were cross-reactive with standard laboratory haplotypes. Evidence supporting the similar genetic organization of wild and standard haplotypes was found, including isolation of separate H-2K and H-2D molecules from a wild-derived strain, and isolation of two separate Ia molecules from a wild-derived strain.     

新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析

利用骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)治疗疾病已经逐渐成为现实,但是作为被移植的种子细胞,BMSCs体外传代能力非常有限,种子细胞来源极为贫乏。本研究通过差速贴壁筛选的方法分离出一种猪BMSCs的衍生细胞株,命名为猪骨髓间充质干细胞衍生细胞(Bone mesenchymal stem-derived cells,BMSDCs)。分别对BMSDCs与BMSCs细胞进行细胞生物学特性分析,探讨其体外诱导分化特性,并应用流式细胞术测定细胞表面标记物。结果表明,BMSC和BMSDCs细胞倍增时间分别为31.3 h和30.3 h,平均传代时间分别为3-5 d和2-3 d;两种细胞均阳性表达CD34、CD90,阴性表达CD44、CD45;经体外诱导后均可分化为成脂细胞和成肌细胞。在传代能力上,前者可传代15至20次,后者可长期传代(200次以上)且维持正常染色体特征。研究认为在适宜的实验条件下,体外培养的猪骨髓间充质干细胞的衍生细胞——BMSDCs能够稳定生存增殖并维持BMSCs多向分化潜能,可作为组织工程的理想种子细胞。     

In Vitro Reconstitution of Anti-Sheep Erythrocyte Antibody Response of T Cell-Depleted Spleen Cells by Allogeneic T Cells or by Factors Derived from Them

Restoration of the impaired antibody response to sheep erythrocytes (SRBC) in cultures of mouse spleen cells, which were deprived of thymus-derived lymphocytes (T cells) by treatment with anti-mouse brain-associated θ (BAθ) antiserum and complement, was studied by adding a small portion of syngeneic or allogeneic normal spleen cells in vitro. Allogeneic spleen cells had a far greater effect than syngeneic spleen cells on the restoration, as far as the normal spleen cells added were able to recognize the alloantigens on the anti-BAθ serum-treated spleen cells (bone marrow-derived lymphocytes). Treatment of the allogeneic spleen cells with mitomycin C did not affect their activity in the restoration of the impaired antibody response. The possibility that the role of T cells in the antibody response to SRBC may be replaced by a nonspecific mediator derived from T cells reacting with allogeneic cells was proven by the finding that supernatant of the mixed allogeneic spleen cell cultures restored the impaired anti-SRBC antibody response of the T cell-depleted spleen cells. The effect of such culture supernatant on the restoration of the antibody response was greatest when it was added to the T cell-depleted spleen cell cultures one day after cultivation with SRBC, suggesting that the effectiveness may result from triggering of the proliferation and differentiation of antibody-forming cell precursors, which have already reacted with the antigen, to antibody-forming cells.     

小鼠骨髓间充质干细胞的分离、培养及鉴定

目的建立一种从小鼠骨髓中分离培养间充质干细胞（MSCs）的高效方法。方法采取贴壁细胞分离法分离和纯化小鼠骨髓间充质干细胞（mMSCs）,检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。结果mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测：CD45、CD11b、CD44及CD29分别为（3.34）%、（2.41）%、（98.46）%及（99.36）%。第4代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。结论通过贴壁培养可以从小鼠骨髓中分离培养出高纯度mMSCs,该方法效率高,稳定性好。     

FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下,成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性;ELISA法测定骨钙蛋白（OC）含量;茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍,Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍,并能形成成熟的羟基磷灰石（HA）结晶和骨结节;FGF-2组的ALP活性比对照组降低了76．7％,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation I. Regeneration and Quantification of Thymocyte Progenitor Cells In the Bone Marrow

Growth kinetics of the donor-type thymus cell population after transplantation of bone marrow into irradiated syngeneic recipient mice is biphasic. During the first rapid phase of regeneration, lasting until day 19 after transplantation, the rate of development of the donor cells is independent of the number of bone marrow cells inoculated. the second slow phase is observed only when low numbers of bone marrow cells (2.5 × 10⁴) are transplanted. the decrease in the rate of development is attributed to an efflux of donor cells from the thymus because, at the same time, the first immunologically competent cells are found in spleen. After bone marrow transplantation the regeneration of thymocyte progenitor cells in the marrow is delayed when compared to regeneration of CFUs. Therefore, regenerating marrow has a greatly reduced capacity to restore the thymus cell population. One week after transplantation of 3 × 10⁶ cells, 1% of normal capacity of bone marrow is found. It is concluded that the regenerating thymus cells population after bone marrow transplantation is composed of the direct progeny of precursor cells in the inoculum.     

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.