Virus-specific immunity after gene therapy in a murine model of severe combined immunodeficiency

Human severe combined immunodeficiency (SCID) can be caused by defects in Janus kinase 3 (JAK3)-dependent cytokine signaling pathways. As a result, patients are at high risk of life-threatening infection. A JAK3 −/− SCID mouse model for the human disease has been used to test whether transplant with retrovirally transduced bone marrow (BM) cells (JAK3 BMT) could restore immunity to an influenza A virus. The immune responses also were compared directly with those for mice transplanted with wild-type BM (+/+ BMT). After infection, approximately 90% of the JAK3 BMT or +/+ BMT mice survived, whereas all of the JAK3 −/− mice died within 29 days. Normal levels of influenza-specific IgG were present in plasma from JAK3 BMT mice at 14 days after respiratory challenge, indicating restoration of B cell function. Influenza-specific CD4⁺ and CD8⁺ T cells were detected in the spleen and lymph nodes, and virus-specific CD8⁺ effectors localized to the lungs of the JAK3 BMT mice. The kinetics of the specific host response correlated with complete clearance of the virus within 2 weeks of the initial exposure. By contrast, the JAK3 −/− mice did not show any evidence of viral immunity and were unable to control this viral pneumonia. Retroviral-mediated JAK3 gene transfer thus restores diverse aspects of cellular and humoral immunity and has obvious potential for human autologous BMT.     

New Strategies for BMT,Organ Transplantation,and Regeneration Therapy

Bone marrow transplantation (BMT) is becoming a powerful strategy for the treatment of hematologic disorders, congenital immunodeficiencies, metabolic disorders and also autoimmune diseases.

We have previously found using various animal models for spontaneous autoimmune diseases, that allogeneic bone marrow transplantation (allo BMT) can be used to prevent and treat various autoimmune diseases. In addition, we have found that autoimmune diseases are stem cell disorders. However, in MRL/lpr mice, which are radiosensitive (<8.5 Gy), we found that conventional BMT had only a transient effect on autoimmune diseases, which were found to recur. Therefore, we concentrated on discovering new strategies to prevent and treat autoimmune diseases in the radiosensitive and chimeric-resistant MRL/lpr mouse.

Using MRL/lpr mice, we established a new method for allo BMT. In this method, whole bone marrow cells (BMCs), containing a small number of T cells and mesenchymal stem cells (MSCs), were directly injected into the bone marrow cavity (intra-bone marrow [IBM]-BMT). MRL/lpr mice treated with IBM-BMT survived more than 2 years without showing the symptoms of autoimmune diseases.

To apply this BMT method to humans, we have also established a new method for BMC harvesting using cynomolgus monkeys. In this method, BMCs are harvested from the long bones using a "Perfusion Method" (PM) and the whole BMCs (including MSCs) are then injected directly into the IBM. We believe that this new method will become a powerful strategy for the treatment of various intractable diseases, including age-associated diseases such as osteoporosis.     

A case of juvenile myelomonocytic leukemia with ocular infiltration

 We present a case of juvenile myelomonocytic leukemia (JMML) with ocular infiltration. A 1-month-old boy presented with myeloid precursors in peripheral blood and a white blood cell count  1 10×10⁹/l. His peripheral blood monocyte count was  1 1×10⁹/l, bone marrow blasts were <20%, and no Ph chromosome was identified. The boy also presented with hepatosplenomegaly, pallor, fever, and skin rash. We diagnosed this case as JMML, although hemoglobin F was within the normal range and no spontaneous colony growth was observed from peripheral blood mononuclear cells. Neither Epstein-Barr (EB) virus nor cytomegalovirus was detected by PCR in bone marrow aspirate or peripheral blood. The patient had several lesions into which JMML cells might have infiltrated, including skin, liver, spleen, oral cavity, right lung, sigmoid colon, and both eyes. To our knowledge, this is the first reported case of JMML with ocular involvement. Since infiltration of JMML cells into both eyes causes blindness, further consideration of the timing of bone marrow transplantation (BMT) in JMML is necessary. Received: February 17, 1999 / Accepted: June 21, 1999     

Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model

It has been suggested that human mesenchymal stem cells (hMSC) could be used to repair numerous injured tissues. We have studied the potential use of hMSC to limit radiation-induced skin lesions. Immunodeficient NOD/SCID mice were locally irradiated to the leg (30 Gy, dose rate 2.7 Gy/min) using a ⁶⁰Co source to induce a severe skin lesion. Cultured bone marrow hMSC were delivered intravenously to the mice. The irradiated skin samples were studied for the presence of the human cells, the severity of the lesions and the healing process. Macroscopic analysis and histology results showed that the lesions were evolving to a less severe degree of radiation dermatitis after hMSC transplant when compared to irradiated non-transplanted controls. Clinical scores for the studied skin parameters of treated mice were significantly improved. A faster healing was observed when compared to untreated mouse. Immunohistology and polymerase chain reaction analysis provided evidence that the human cells were found in the irradiated area. These results suggest a possible use of hMSC for the treatment of the early phase of the cutaneous radiation syndrome. A successful transplant of stem cells and subsequent reduction in radiation-induced complication may open the road to completely new strategies in cutaneous radiation syndrome therapy.     

Graft-versus-leukaemia activity can be predicted by natural cytotoxicity against leukaemia cells

We have investigated graft-versus-leukaemia (GVL) effects after allogeneic bone marrow transplantation (BMT), using three murine leukaemia models, A20 (B lymphocytic), WEHI-3 (myelomonocytic) and PU5-1R (myeloid). Injection of leukaemia cells in a high number (10⁶ cells) into syngeneic Balb/c mice (H-2^d) invariably led to death with a median survival time of 22 d (A20), 18 d (WEHI-3) and 45 d (PU5-1R). A lower tumour load of A20 (5 × 10⁵ cells) was used in some experiments resulting in a leukaemic death rate of 94%. Lethal total-body irradiation followed by syngeneic BMT prolonged survival (P < 0.05) for animals bearing the leukaemia A20 and WEHI-3 but was unsuccessful for animals injected with cells from the monocytic leukaemia PU5-1R. Graft-versus-host (GVH)-nonreactive marrow of (C57 × Balb/c)F1 mice (H2^bxd) exerted a significant GVL-effect with reduced relapse rate and improved survival in mice receiving the leukaemia cell line A20. In animals with low tumour load a significant reduction of the relapse rate from 82% following syngeneic BMT to 47% following allogeneic, GVH-nonreactive BMT could be achieved. Depletion of natural killer (NK) cells from the GVL-reactive semi-allogenic bone marrow graft enhances the relapse rate of the leukaemia A20 to 65%. In mice bearing the leukaemias WEHI-3 or PU5-1R allogeneic GVH-nonreactive BMT did not improve survival compared to syngeneic BMT. Transplantation of GVH-reactive bone marrow from DBA mice (MHC identical to Balb/c, minor difference) caused only a limited and insignificant reduction of relapse rate for animals with the leukaemia A20. These in vivo data are in close correlation with in vitro natural killer cell (NK) activity of the donor strains against the respective leukaemia targets. Depletion of NK cells from the GVL-reactive (C57 × Balb/c)F1 bone marrow resulted in a significant loss of GVL activity. We conclude that NK cells are involved in graft-versus-leukaemia effects independent of graft-versus-host disease (GVHD).     

New strategies for BMT,organ transplantation,and regeneration therapy

Bone marrow transplantation (BMT) is becoming a powerful strategy for the treatment of hematologic disorders, congenital immunodeficiencies, metabolic disorders and also autoimmune diseases. We have previously found using various animal models for spontaneous autoimmune diseases, that allogeneic bone marrow transplantation (allo BMT) can be used to prevent and treat various autoimmune diseases. In addition, we have found that autoimmune diseases are stem cell disorders. However, in MRL/lpr mice, which are radiosensitive (<8.5 Gy), we found that conventional BMT had only a transient effect on autoimmune diseases, which were found to recur. Therefore, we concentrated on discovering new strategies to prevent and treat autoimmune diseases in the radiosensitive and chimeric-resistant MRL/lpr mouse. Using MRL/lpr mice, we established a new method for allo BMT. In this method, whole bone marrow cells (BMCs), containing a small number of T cells and mesenchymal stem cells (MSCs), were directly injected into the bone marrow cavity (intra-bone marrow [IBM]-BMT). MRL/lpr mice treated with IBM-BMT survived more than 2 years without showing the symptoms of autoimmune diseases. To apply this BMT method to humans, we have also established a new method for BMC harvesting using cynomolgus monkeys. In this method, BMCs are harvested from the long bones using a "Perfusion Method" (PM) and the whole BMCs (including MSCs) are then injected directly into the IBM. We believe that this new method will become a powerful strategy for the treatment of various intractable diseases, including age-associated diseases such as osteoporosis.     

Multipotential hematopoietic stem cells in the bone marrow

It has been generally held that human hematopoietic stem cells are lineage-negative CD34⁺ CD38^?. However, murine hematopoietic stem cells were reported to be CD34^?. We have characterized the surface phenotypes of murine hematopoietic stem cells by using a murine transplantation model. Our studies revealed that the majority of the stem cells in normal adult mice are CD34^? while a minority (15%–20%) being CD34⁺. Our studies also revealed that stem cells that are activated by injection of 5-fluorouracil in vivo, exposure to cytokines in vitro, or mobilization by G-CSF are CD34⁺ and that CD34 expression is reversible. It has been reported that fetal murine hematopoietic stem cells are CD34⁺. Our studies revealed that stem cells of juvenile mice are CD34+ and that the developmental change from CD34⁺ to CD34^? state takes place between 7 and 10 weeks of age. In adult mice, expression of CD38 by steady-state and activated stem cells was completely reciprocal of CD34 expression. Activated stem cells and the minority population of the stem cells in the normal mice are CD34⁺ CD38^?. In contrast, the majority of stem cells in normal adult mice are CD34- CD38+. Recently, we studied CD38 expression by stem cells of neonatal and juvenile mice. Stem cells of newborn mice are CD38^?. About half of the stem cells of 5-week-old mice are CD38⁺. Finally, our studies indicated that some of the CD34⁺ stem cells in the bone marrow of normal adult mice express lineage markers such as Mac-1 and CD4. These studies in a murine model clearly documented that expression of both CD34 and CD38 by stem cells is under developmental control and may be subject to changes induced by activation of the stem cells. In order to test whether or not these principles apply to human stem cells we tested surface phenotypes of human stem cells using two xenotransplantation techniques. Studies based on human/sheep xenograft model indicated that a significant portion of adult human long-term engrafting cells are CD34^?. Similar to mouse stem cells expression of CD34 by human stem cells was reversible. Studies based on our newborn NOD/SCID/β-microglobulin^null mice indicated that human cord blood stem cells are CD34⁺ CD38^?. These results appear to support the validity of studies of murine stem cells to provide insight into human stem cells.     

New strategies for allogeneic bone marrow transplantation and organ allografts

In humans, the success rate of bone marrow transplantation (BMT) across major histocompatibility complex (MHC) barriers is not high due to: (1) graft-versus-host reaction (GvHR); (2) graft rejection, and (3) incomplete T cell recovery. In mice, GvHR can be prevented if T cell- depleted bone marrow cells (BMCs; <2% T cells) are used. Graft rejection can be prevented by either bone grafts (to recruit donor-derived stromal cells) or the injection of donor BMCs via the portal vein (p.v; to induce donor-specific tolerance). T cell functions are recovered by BMT plus bone grafts if the thymic functions of recipients are not completely lost. After the complete loss of thymic functions (due to aging), BMT plus embryonal thymus grafts should be carried out.Recently, we have found that persistent donor-specific tolerance can be induced if allogeneic hemopoietic stem cells are injected via the p.v. Based on these findings, we have established new strategies for organ allografts. Without irradiation, donor BMCs should be injected from the p.v. injection on day 0 plus i.v. injection on day 5, and an immunosuppressant (CsA or FK506) should be used on days 2 and 5. Without using immunosuppressants, sublethal irradiation (7 Gy) followed by skin allografts plus allogeneic BMC injection via the p.v. should be carried out. This leads to a 100% acceptance of skin and pancreas allografts for more than 300 days. The recipient mice show mixed allogeneic chimerism, and spleen cells from the recipients show tolerance to both donor-type and host-type MHC determinants in the assays for mixed lymphocyte reaction and generation of cytotoxic T lymphocytes. We have confirmed that these strategies are applicable to other animals such as pigs and rats. We therefore believe that they will become viable and valuable strategies for human organ allografts.     

Bone marrow-derived keratinocytes are not detected in normal skin and only rarely detected in wounded skin in two different murine models

OBJECTIVE: Because the ability of bone marrow-derived cells (BMDCs) to repopulate tissues and the possible mechanisms of repopulation remain controversial, we used two distinct murine models to determine whether BMDCs can repopulate epidermal keratinocytes during either steady-state homeostasis or after tissue injury. METHODS: The accessibility of skin keratinocytes makes it an excellent tissue to assess BMDC repopulation. In the two murine models, BMDCs from either male homologous B6, 129S Rosa26 mice that constitutively express ss-galactosidase or male hemizygote C57 BL/6-Tg(ACTbEGFP)1Osb/J mice expressing enhanced green fluorescent protein were transplanted via tail vein injection into control lethally irradiated (9.5 Gy) congenic female recipients and the percentage of keratinocytes derived from the transplanted BMDCs, both with and without wounding, was carefully determined. RESULTS: Analysis of bone marrow, thymus, spleen, and lymph nodes confirmed complete engraftment of donor BMDCs 6 months post-bone marrow transplantation. However, during steady-state homeostasis, bone marrow-derived keratinocytes could not be detected in the epidermis. In a skin wound-healing model, the epidermis contained only rare bone marrow-derived keratinocytes (< 0.0001%) but did contain scattered bone marrow-derived Langerhans cells. CONCLUSIONS: These results suggest that BMDCs do not significantly contribute to steady-state epidermal homeostasis and are not required or responsible for providing keratinocyte stem cells and keratinocyte repopulation following skin injury.     

Turning human epidermis into pancreatic endoderm

OBJECTIVE: Human embryonic stem (hES) cells can be differentiated into pancreatic endoderm structures in vitro. The study was performed to determine whether induced pluripotent stem (iPS) cells can be differentiated into similar structures with comparable efficiency. METHODS: We compared the ability of hES cells and iPS cells derived from human epidermal keratinocytes to progressively differentiate into pancreatic endoderm. Human foreskin keratinocytes were reprogrammed to pluripotency by transduction with retroviruses encoding Oct4, Sox2, and Klf4. The resulting keratinocyte-derived iPS (KiPS) cell lines and a hES cell line were subjected to a modified pancreatic endoderm differentiation protocol. Cells and embryoid-body structures derived from both hES and KiPS cells were compared at different stages of development for expression of stem cell and differentiation markers, including Sox2, Oct4, Mixl1, Brachyury, Gsc, FoxA2, Sox17, Hnf4α, Hnf1β, Nkx2.2, Nkx6.1, Hex, Isl1, Pdx1, and Slc2A, via Taqman real-time PCR, flow-cytometry, and/or immunocytochemistry. RESULTS: hES cells and KiPS cells expressed similar levels of the stem cell factors Sox2 and Oct4. Upon differentiation, both cell types underwent remarkably similar changes in gene expression. They acquired the definitive endoderm markers Sox17 and FoxA2. Most Sox17⁺ and FoxA2⁺ cells co-expressed Hnf4α and Hnf1β, found in the primitive gut tube, a pancreas precursor. Most FoxA2⁺ cells were also Pdx1⁺, and many expressed Nkx2.2, Nkx6.1, and Isl1. CONCLUSIONS: Keratinocyte-derived iPS cells can be differentiated into pancreatic endoderm, and the efficiency of this process is comparable to that seen for hES cells. Thus keratinocytes have the potential to serve as a source of patient-specific pancreatic endoderm for transplantation.     

Treatment of autoimmune diseases by hematopoietic stem cell transplantation

Remarkable advances have been made in bone marrow transplantation (BMT), which now has become a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency disorders, and autoimmune diseases. Using various animal models, allogeneic BMT has been found to be useful in the treatment of autoimmune diseases. In MRL/lpr mice, which are radiosensitive (<8.5 Gy) and are an animal model for autoimmune disorders, conventional BMT resulted in only transient effects; the manifestations of the autoimmune diseases recurred 3 months after BMT. However, the combination of BMT plus bone grafts (to recruit donor stromal cells) was capable of preventing the recurrence of autoimmune diseases in MRL/lpr mice. This strategy was found to be ineffective in the treatment of MRL/lpr mice that had developed autoimmune diseases, because these mice were more sensitive to the effects of radiation after the onset of lupus nephritis due to uremic enterocolitis. We have recently discovered a safer strategy for treatment of autoimmune diseases, which includes fractionated irradiation (5.5 Gy x 2) (day -1) followed by portal venous injection (day 0) plus intravenous injection (day 5) of donor unfractionated bone marrow cells. We successfully treated autoimmune diseases in MRL/lpr mice using this strategy; 100% of MRL/lpr mice treated in this fashion survive >1 year after treatment. We identified the mechanisms underlying the components of this approach and have found that stromal cells play a crucial role in successful BMT. In this review, the conditions essential for successful allogeneic BMT are discussed.     

Osteopontin Promotes Left Ventricular Diastolic Dysfunction Through a Mitochondrial Pathway

BackgroundPatients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction, and kidney failure in Col4a3^−/− mice, a model of human Alport syndrome.ObjectivesThe purpose of this study was to identify potential OPN targets in biopsies of HF patients, healthy control subjects, and human induced pluripotent stem cell–derived cardiomyocytes (hiPS-CMs), and to characterize the cardiac phenotype of Col4a3^−/− mice, relate this to HFpEF, and investigate possible causative roles for OPN in driving the cardiomyopathy.MethodsOGDHL mRNA and protein were quantified in myocardial samples from patients with HFpEF, heart failure with reduced ejection fraction, and donor control subjects. OGDHL expression was quantified in hiPS-CMs treated with or without anti-OPN antibody. Cardiac parameters were evaluated in Col4a3^−/− mice with and without global OPN knockout or AAV9-mediated delivery of 2-oxoglutarate dehydrogenase-like (Ogdhl) to the heart.ResultsOGDHL mRNA and protein displayed abnormal abundances in cardiac biopsies of HFpEF (n = 17) compared with donor control subjects (n = 12; p < 0.01) or heart failure with reduced ejection fraction patients (n = 12; p < 0.05). Blockade of OPN in hiPS-CMs conferred increased OGDHL expression. Col4a3^−/− mice demonstrated cardiomyopathy with similarities to HFpEF, including diastolic dysfunction, cardiac hypertrophy and fibrosis, pulmonary edema, and impaired mitochondrial function. The cardiomyopathy was ameliorated by Opn^−/− coincident with improved renal function and increased expression of Ogdhl. Heart-specific overexpression of Ogdhl in Col4a3^−/− mice also improved cardiac function and cardiomyocyte energy state.ConclusionsCol4a3^−/− mice present a model of HFpEF secondary to CKD wherein OPN and OGDHL are intermediates, and possibly therapeutic targets.     

Interleukin-7 improves T-cell recovery after experimental T-cell-depleted bone marrow transplantation in T-cell-deficient mice by strong expansion of recent thymic emigrants

Interleukin-7 (IL-7) has been shown to enhance thymic output of newly developed T cells following bone marrow transplantation (BMT) in mice. In addition, IL-7 may affect peripheral expansion of T cells. In order to study the relative contribution of thymopoiesis versus peripheral T-cell expansion in the setting of compromised thymopoiesis, we have applied IL-7 in an experimental stem cell transplantation model using T cell-deficient RAG-1(-/-) mice. C57BL/6 RAG-1(-/-) mice received transplants of syngeneic T-cell-depleted (TCD) bone marrow (Ly5.1) with or without supplemented T cells (Ly5.2). IL-7 was administered until day 63 after BMT. Peripheral blood T- and B-cell recovery was quantified by flow cytometry and thymopoiesis was studied by quantification of T-cell receptor rearrangement excision circles (TRECs). In mice receiving a T-cell-replete BMT, IL-7 selectively expanded mature CD45.2+ T cells without affecting the recovery of new bone marrow-derived CD45.1+ T cells. In contrast, IL-7 significantly enhanced the recovery of bone marrow-derived T cells after TCD BMT. Quantification of TRECs in mice receiving a TCD BMT revealed that enhanced T-cell recovery following IL-7 treatment resulted from a strong expansion of newly developed naive T cells. These results suggest that peripheral expansion of recent thymic emigrants or mature T cells may be a preferential mechanism by which IL-7 enhances T-cell recovery after BMT.     

干细胞因子在骨髓干细胞心肌内移植治疗中的作用研究

目的探讨干细胞因子（SCF）在骨髓干细胞心肌内移植中的作用机制及对移植治疗的影响。方法对急性心肌梗死（AMI）大鼠进行骨髓干细胞心肌内移植治疗,RT—PCR方法检测骨髓中SCF表达水平的变化,并用SCF表达水平不同的骨髓干细胞进行心肌内移植治疗,超声心动图检测心脏结构、功能的变化;ELISA方法检测心肌组织、血和骨髓中SCF蛋白水平的变化。结果AMI1周时骨髓中SCF mRNA表达明显降低（P〈0．01）,接受移植治疗大鼠骨髓中SCF mRNA表达未出现降低。不同SCF表达水平的骨髓干细胞进行心肌内移植治疗均能使心脏射血分数达到70％,各组之间差异无统计学意义。移植治疗后1周血清中SCF蛋白的表达水平明显低于正常对照（P〈0．05）。结论骨髓干细胞心肌内移植可能通过SCF产生骨髓动员作用;心肌梗死或曾接受移植治疗个体的骨髓干细胞同样可用于心肌内移植治疗,不影响心功能的改善。     

Immunoregulatory effects of allogeneic mixed chimerism induced by nonmyeloablative bone marrow transplantation on chronic inflammatory arthritis and autoimmunity in interleukin‐1 receptor antagonist–deficient mice

Objective

To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T cell–depleted, nonmyeloablative bone marrow transplantation (BMT) on chronic inflammatory arthritis and autoimmunity in mice deficient in interleukin‐1 receptor antagonist (IL‐1Ra).

Methods

IL‐1Ra^−/− mice (H‐2K^d) were treated with antibody to asialoganglioside G_M1 (anti–natural killer cell), total body irradiation (500 cGy), and T cell–depleted, nonmyeloablative BMT derived from C57BL/6 mice (H‐2K^b). Engraftment and chimerism were evaluated in peripheral blood, lymph nodes, and spleen by multicolor flow cytometry. The severity of arthritis was evaluated by clinical scoring and histopathologic assessment. Levels of IgG1 and IgG2a subtypes of anti–type II collagen (anti‐CII) antibodies were measured in serum samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T cell proliferative responses and levels of cytokine production (interferon‐γ [IFNγ], tumor necrosis factor α [TNFα], interleukin‐10 [IL‐10], and IL‐17) were assayed in culture supernatants.

Results

All IL‐1Ra^−/− mice receiving BMT showed marked improvement in arthritis within 3 weeks, as well as successful induction of mixed chimerism. These mice showed higher levels of IgG1, and lower levels of IgG2a anti‐CII antibodies and weaker T cell proliferative responses than did mice in the control groups (either no treatment or conditioning alone without bone marrow rescue). In mixed chimeras, the levels of IFNγ, TNFα, and IL‐17 produced from CII‐stimulated T cells were significantly suppressed and IL‐10 production was significantly higher as compared with controls.

Conclusion

The introduction of allogeneic mixed chimerism showed a strong immunoregulatory potential to correct established chronic inflammatory arthritis and autoimmunity originating from a dysregulated proinflammatory cytokine network.

     

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)‐mediated immunosuppression involves TGF‐β and indoleamine 2,3‐dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft‐versus‐host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC‐treated group, lethally irradiated DBA/2(H‐2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H‐2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC‐treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.     

Cellular and cytokine effectors of acute graft versus host disease

Graft-versus-host-disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT) and Older BMT recipients are at greater risk for acute graft-versus-host-disease. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older recipients. GVHD mortality and morbidity, as well as pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice. In a haploidential GVHD model, CD4⁺ donor T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. We also evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD in mouse models. In bone marrow chimeras recipients in which either MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GVHD. These results suggest new strategies for the prevention and treatment of this toxic complication of BMT.     

Intrauterine transplantation of human fetal mesenchymal stem cells from first-trimester blood repairs bone and reduces fractures in osteogenesis imperfecta mice

The inherited skeletal dysplasia osteogenesis imperfecta (OI) results in multiple fractures and is currently treated empirically. We transplanted human first-trimester fetal blood mesenchymal stem cells (MSCs) into homozygous oim mice in utero. This resulted in a two-thirds reduction in long bone fractures (P < .01), with fewer fractures per mouse (median 1, range 0-2 in mice that received transplants vs median 3, range 1-5 in mice that did not receive transplants by 12 weeks, P < .01). Nearly all mice that did not receive transplants had fractures (47 [97.9%] of 48), in contrast to 17 (58.6%) of 29 4- to 12-week-old mice that received transplants (P < .01). Transplantation was associated with increased bone strength (P < .01), thickness (P < .01), and length (P < .01), and normalization/reduction of growth plate height in 4- to 12-week-old oim was reduced in mice that underwent transplantion (P < .001). More donor cells were found in bone tissues compared with other organs (P < .001), with cells clustered in areas of active bone formation and remodeling, and at sites of fracture healing. Donor cells found in the bone expressed osteoblast lineage genes, and produced the extracellular bone structural protein osteopontin. Finally, MSC transplantation decreased bone hydroxyproline content. In conclusion, intrauterine transplantation of fetal MSCs markedly reduced fracture rates and skeletal abnormalities in a mouse model of the intermediate severity type III OI, suggesting a scientific basis for MSC treatment of affected human fetuses.     

Clinical and prognostic significance of the Philadelphia chromosome in adult patients with acute lymphoblastic leukemia

Summary Between 1983 and 1991 the Philadelphia chromosome (Ph¹) was found in bone marrow and/or peripheral blood cells of 25 adult patients with acute lymphoblastic leukemia (ALL). The Ph¹ as sole anomaly was seen in 13 patients, while six patients had additional structural and another six structural and numerical aberrations. Most patients (23/25) received combination chemotherapy according to the BMFT protocols 1/81, 2/84, 3/87, and 4/89. For 25 evaluable patients two early deaths, two treatment failures, two partial remissions (PR), and 19 complete remissions (CR) after phase 1 or 2 of the induction regimen were recorded. Two of these 19 patients who achieved CR are presently disease free, whereas 17 have relapsed after a median duration of remission of 9 months. Actuarial median survival for all patients was 13 months. The probability of continuous complete remission (CCR) after 39 months, as well as that of survival after 40 months, is only 6%. Our results confirm that the presence of the Ph¹ is associated with a poor prognosis in adult-ALL patients. Therefore, whenever first CR is obtained and an HLA-identical donor is available, allogeneic bone marrow transplantation (BMT) should be performed at once, the more so, since transplantation in second CR seems to offer no cure. Future studies will have to show whether an intensified cytotoxic therapy can improve the prognosis of Ph¹⁺-ALL.     

Therapeutic effects of induced pluripotent stem cells in chimeric mice with β-thalassemia

Although β-thalassemia is one of the most common human genetic diseases, there is still no effective treatment other than bone marrow transplantation. Induced pluripotent stem cells have been considered good candidates for the future repair or replacement of malfunctioning organs. As a basis for developing transgenic induced pluripotent stem cell therapies for thalassemia, β⁶⁵⁴ induced pluripotent stem cells from a β⁶⁵⁴ -thalassemia mouse transduced with the normal human β-globin gene, and the induced pluripotent stem cells with an erythroid-expressing reporter GFP were used to produce chimeric mice. Using these chimera models, we investigated changes in various pathological indices including hematologic parameters and tissue pathology. Our data showed that when the chimerism of β⁶⁵⁴ induced pluripotent stem cells with the normal human β-globin gene in β⁶⁵⁴ mice is over 30%, the pathology of anemia appeared to be reversed, while chimerism ranging from 8% to 16% provided little improvement in the typical β-thalassemia phenotype. Effective alleviation of thalassemia-related phenotypes was observed when chimerism with the induced pluripotent stem cells owning the erythroid-expressing reporter GFP in β⁶⁵⁴ mouse was greater than 10%. Thus, 10% or more expression of the exogenous normal β-globin gene reduces the degree of anemia in our β-thalassemia mouse model, whereas treatment with β⁶⁵⁴ induced pluripotent stem cells which had the normal human β-globin gene had stable therapeutic effects but in a more dose-dependent manner.