Spontaneous degenerative polyarthritis in male New Zealand black/KN mice

Histopathologic studies and radiographic analysis revealed that male New Zealand black/KN (NZB/KN) mice develop degenerative polyarthritis in the joints of the forepaw and hindpaw beginning at age 2 months. Deposits of autoantibodies were observed on proliferating collagen fibers, nuclei of chondrocytes, and epidermal cells. Increases in the frequency of positivity for rheumatoid factor and anti-type II collagen antibodies and in the level of serum oxidation activity were noted in these mice. The joint disease in male NZB/KN mice was transferable to female NZB/KN mice and male BALB/c mice by intraperitoneal injection of spleen cells from the male NZB/KN mice. This animal model of arthritis will be extremely useful for analyzing not only the pathogenesis of rheumatoid arthritis, but also new strategies for its treatment, since NZB/KN mice, unlike MRL/lpr mice, do not develop severe lupus nephritis or lymph-adenopathy, and therefore have a longer survival period.     

Altered bone marrow cell sensitivity in the lupus-prone NZB/W mouse: regulation of CFU-GM colony formation by estrogen, tamoxifen and thrombopoietin

Estrogen is thought to contribute to the onset of systemic lupus erythematosus (SLE) in women through mechanisms that are not completely understood. Although estrogen serves as a negative regulator in normal hematopoietic development, little research has been conducted examining alteration in hematopoietic development triggered by estrogen in lupus-susceptible individuals. We examined whether estrogen and other factors could influence colony formation of bone marrow cells obtained from normal and lupus-susceptible mice. Bone marrow cells isolated from New Zealand Black (NZB) and lupus-prone New Zealand Black and New Zealand White cross (NZB/W) mice were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) alone or in combination with estrogen, thrombopoietin (TPO), tamoxifen, estrogen and TPO. or estrogen and tamoxifen, and plated in methylcellulose culture medium. Plates were scored for the number of CFU-GM (colony forming unit granulocyte-macrophage) colonies after 6d in culture. For females of both mouse strains, estrogen significantly decreased (P < 0.05) the number of GM colonies. Co-treatment of NZB/W cells, but not NZB cells, with TPO or tamoxifen reversed the suppressive action of estrogen (P < 0.05). In contrast, while estrogen did suppress colony formation from cells of NZB/W males (P < 0.05), neither TPO nor tamoxifen reversed this effect. Our results indicate that the sensitivity of bone marrow cells isolated from both female and male NZB/W lupus-prone mice to hormones/growth factors is qualitatively different from cells of NZB mice, and suggest that hematopoietic alterations at the level of the bone marrow may be related to the pathogenesis of SLE.     

A preliminary study of liver cells derived from bone marrow

OBJECTIVE: To investigate whether hepatocytes can be derived from bone marrow cells in vivo. METHODS: A cohort of lethally irradiated BALB/C female mice received whole bone marrow transplants from age‐matched male donors and were killed at 1, 2, 4 and 8 weeks post transplantation. Fluorescence in situ hybridization (FISH) for the Y‐chromosome was performed using liver tissue. RESULTS: A few Y‐chromosome positive hepatocytes were found in the liver tissues at 2 months post transplantation. CONCLUSION: Hepatocytes can be derived from bone marrow cells after transplantation in lethally irradiated mice without severe acute hepatic injury.     

Transplantation of wheat germ agglutinin-positive hematopoietic cells to prevent or induce systemic autoimmune disease.

Hematopoietic stem cell defects are thought to be involved in the etiopathogenesis of systemic autoimmune disease. Positively selected, stem cell-enriched populations of wheat germ agglutinin-positive (WGA+) low-density bone marrow and fetal liver cells from normal and autoimmune-prone mice were used to determine whether reciprocal transplantation of stem cells between normal and autoimmune-prone mice inhibits or causes development of autoimmune disease. NZB recipients of DBA/2 stem cell populations analyzed greater than 100 days after bone marrow or fetal liver cell transplantation showed decreased levels of anti-DNA antibodies and decreased glomerular lesions when compared with nontreated NZB mice or NZB recipients of NZB stem cell preparations. Female DBA/2 recipients of WGA+ NZB bone marrow cell or fetal liver cell transplants exhibited elevated serum autoantibody levels and developed glomerular lesions characteristic of NZB mice when analyzed greater than 100 days after transplantation. These pathological disturbances were not observed in DBA/2 recipients of DBA/2 stem cell preparations. The data indicate that WGA+ stem cells from autoimmune-prone NZB mice contain the genetic defects responsible for the development of systemic autoimmune disease.     

B220- bone marrow progenitor cells from New Zealand black autoimmune mice exhibit an age-associated decline in Pre-B and B-cell generation

New Zealand Black (NZB) autoimmune mice exhibit progressive, age- dependent reduction in bone marrow pre-B cells. To ascertain the capacity of NZB bone marrow B220- cells to generate pre-B cells in a supportive environment, B-lineage (B220+) cell-depleted and T-cell- depleted bone marrow cells from NZB mice at 1 to 3, 6, and 10 to 11 months of age were adoptively transferred into irradiated (200R) C.B17 severe combined immunodeficient (SCID) mice. Bone marrow pre-B cells (sIgM- CD43[S7]- B220+) were assessed 3 and 10 weeks posttransfer. Pre- B cells and B cells were reconstituted in SCID recipients of older NZB progenitor cells by 10 weeks posttransplant, in contrast to the very low numbers of pre-B cells present in the donor bone marrow. However, B220- bone marrow progenitor cells from greater than 10-month-old NZB donors were deficient in the reconstitution of both pre-B and B cells in SCID recipients at 3 weeks post-transfer. This reflected a slower kinetics of repopulation, because older NZB-->SCID recipients had numbers of both pre-B and B cells similar to recipients of young NZB progenitor cells by 10 weeks posttransplant. Adoptive transfer of equal mixtures of BALB/c and older NZB bone marrow B220- progenitor cells into irradiated C.B17 SCID recipients failed to demonstrate active suppression. These results suggest that, with age, NZB bone marrow has reduced numbers and/or function of early B220- B-lineage progenitors. Consistent with this hypothesis, B220- bone marrow cells from older NZB mice were deficient in progenitors capable of yielding interleukin-7 (IL-7) responsive pre-B cells in vitro on stimulation with the pre-B- cell potentiating factor, insulin-like growth factor 1 (IGF-1).     

Altered Bax expression and decreased apoptosis in bone marrow cells of lupus-susceptible NZB/W mice.

Estrogen is believed to contribute to the development of the autoimmune disorder systemic lupus erythematosus (SLE) (lupus) in women. We hypothesized that estrogen might promote the development of lupus by altering apoptosis of bone marrow cells, perhaps through regulation of the apoptotic proteins Bax and Bcl-2. We compared the effects of estrogen (E2) and thrombopoietin (TPO) on the expression of Bax or Bcl-2 in bone marrow cells isolated from female non-lupus (NZW or NZB parental strains) or lupus-prone (NZB and NZW cross; NZB/W) mice. We report that the basal level of Bax in parental bone marrow cells was significantly greater than that of cells from NZB/W animals. Treatment of NZB or NZW marrow cells with E2 resulted in a significant decrease in Bax expression, which was completely reversed upon co-treatment with TPO. Bax expression was not significantly altered by E2 and/or TPO in NZB/W cells. Bcl-2 levels did not differ between murine strains under basal or hormone-treated conditions. Lower basal expression of Bax protein was associated with significantly less apoptosis for NZB/W marrow cells. In addition, there were significantly greater numbers of cells in bone marrow of lupus-susceptible animals. Our results indicate that bone marrow cells of NZB/W animals differ physiologically from those of NZW or NZB mice, and suggest that decreased expression of Bax in bone marrow precursors may lead to decreased apoptosis of mature blood cells in lupus-susceptible mice.     

Mesenteric hemopoietic colonies. II. Occurrence in mice after transplantation of syngeneic normal bone marrow cells.

Syngeneic normal bone marrow cells (BMC) were i.p. transplanted into six different strains of lethally irradiated mice (other than BALB/c). The occurrence of mesenteric hemopoietic colonies (MHC) was determined microscopically. The size and numbers of MHC were augmented when the transplantation period was lengthened. On the tenth day spleen colonies were found in all mice.     

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells).Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction.These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.     

Effect of estrogen on natural killer cells

Treatment of mice with sustained high levels of β-estradiol leads to a reduction in natural killer cell activity and genetic resistance to bone marrow transplantation. The loss of natural killing does not seem to result from either humoral or immune suppression. Natural killer cells are thought to depend on the bone marrow, and it is notable that estrogens reduce natural killing at approximately the same time that they produce a loss of marrow due to osteoproliferation. Similarly, mice with congenital osteopetrosis are deficient in natural killing. However, changes in natural killing during and after treatment with estrogen do not correspond directly to changes in marrow volume. Estrogens are known to exacerbate spontaneous autoimmunity in NZB/NZW mice. The relationship between this effect and the effect of estrogen on natural killing is not clear. When natural killing is lowered in NZB/NZW mice by the in vivo administration of ⁸⁹Sr, autoimmunity is reduced.     

Correction of copper metabolism is not sustained long term in Wilson’s disease mice post bone marrow transplantation

Purpose Alternative cell sources have been sought for the treatment of liver diseases, since liver cells are in short supply for cell transplantation. Although bone marrow-derived cells have been investigated as an alternative cell source, few studies have demonstrated long-term disease correction. Here we examined bone marrow stem cell transplantation into the toxic milk (tx) mouse model for Wilson’s disease, a mild liver disease characterized by hepatic copper accumulation. The aim of this study was to determine whether bone marrow cells engrafted in the liver could sustain correction of abnormal copper metabolism in the tx mouse. Methods Bone marrow cells were isolated from congenic normal mice, transduced to express enhanced green fluorescent protein, sorted for stem cell (Sca-1) and lineage cell (Lin) surface markers, and then transplanted into sub-lethally irradiated normal or tx mice. Analysis for donor cell activity and distribution was undertaken 5 and 9 months post-transplant to allow for sufficient time to repopulate the liver and demonstrate disease correction. Results Donor bone marrow cells engrafted in both normal and tx mouse liver within 5 months. Significantly reduced liver copper was found in tx mice with donor cell liver engraftment at 5 months post-transplant compared to controls, demonstrating partial correction of abnormal copper metabolism in the short term. However, disease correction was not maintained 9 months post-transplantation. Lin⁻Sca-1⁺ cells were more likely to partially correct disease than Lin⁺Sca-1⁻ cells in the short term. Conclusion These results demonstrate that bone marrow transplants cannot maintain disease correction in a mouse model of mild hepatic damage, although initial copper metabolism correction was observed.     

Accelerated platelet reconstitution following transplantation of bone marrow cells derived from IL-6-treated donor mice

 We transplanted bone marrow cells derived from normal donor mice treated with IL-6 to study the effect on the hematopoietic recovery of lethally irradiated (8.5 Gy) recipients. Male Balb/C mice were treated for 7 days by continuous infusion of IL-6 (10 μg/day). Not only did these donor mice have increased numbers of circulating platelets as was previously shown; the numbers of circulating progenitor cells also increased more than 25-fold. Transplantation of nucleated bone marrow cells derived from these donor mice into lethally irradiated female recipients resulted in increased platelet nadir counts in comparison to recipients of normal bone marrow cells and similar to nadir counts of recipients of normal donor bone marrow treated with IL-6 for 7 days after transplantation. Combination of transplantation of bone marrow derived from IL-6 treated donors with post-transplantation treatment of the recipients with IL-6 resulted in a further increase in nadir counts, although it did not cause a further acceleration of platelet reconstitution. We conclude that transplantation of bone marrow cells modified in vivo by IL-6 results in significantly accelerated reconstitution of platelets, to a degree similar to that observed following treatment with IL-6 after transplantation. Received: 6 June 1996 / Accepted: 3 July 1996     

Impaired ability of bone marrow cells from immunodeficient mice to establish long-term cultures

Summary Haemopoiesis is often depressed in patients suffering from acquired immune deficiency syndrome (AIDS). Although several mechanisms have been postulated to be responsible for depressed haemopoiesis in AIDS patients, the aetiology of this disorder is still unknown. We hypothesized that failure of the stromal microenvironment may account for part of the haemopoietic defect observed in patients with AIDS. We therefore studied a murine model of AIDS (MAIDS) caused by infection with LP-BM5 virus to determine the ability of bone marrow cells from immunodeficient mice to establish long-term stromal cultures. In addition, normal and MAIDS mice received AZT (2 mg/ml) in their drinking water for up to 1 month to determine the effects of AZT treatment in vivo on the ability of bone marrow cells to support haemopoiesis in long-term cultures. Decreased numbers of non-adherent cells were observed in long-term bone marrow cultures (LTBMC) of MAIDS mice when compared to cultures derived from normal mice. Decreased numbers of non-adherent cells were observed in cultures of bone marrow cells from AZT-treated normal mice, when compared to untreated normal controls. Cells from AZT-treated MAIDS mice produced the smallest number of non-adherent cells. BFU-E and CFU-G/M were decreased in cultures of MAIDS mice when compared to those of normal mice. AZT-treatment further decreased the number of colony-forming cells in both MAIDS and normal cultures. Stromal cell function of MAIDS mice was also assessed by inoculating non-adherent cells from normal mice onto confluent irradiated MAIDS LTBMC. Stroma from MAIDS mice was unable to support haemopoietic function of normal bone marrow cells. Polymerase chain reaction (PCR) analysis of steady state levels of cytokine mRNAs of cells from confluent cultures revealed that levels of interleukin-6 mRNA were unchanged in MAIDS mice, as compared to normal controls, but the levels of GM-CSF were decreased in MAIDS mice. These data suggest that LP-BM5 MuLV infection alters the functioning of the haemopoietic stroma and that one mechanism of this depression in haemopoiesis may be via alterations of cytokine production.     

In vivo imaging studies of the effect of recipient conditioning,donor cell phenotype and antigen disparity on homing of haematopoietic cells to the bone marrow

Summary. Homing of transplanted bone marrow cells (BMC) to the host bone marrow (BM) is the first step of engraftment towards durable multilineage haematopoietic reconstitution. We used an in vivo assay to track PKH‐labelled cells in the BM of mice after transplantation, using fluorescence microscopy through an optical window placed over the distal femoral epiphysis. Within hours after intravenous injection, the cells moved in and out the femur, and were mobile within the marrow space. One hour after injection of whole BMC into non‐conditioned syngeneic and allogeneic recipients, the homing efficiencies (HE) were 1·23 ± 0·14% and 0·12 ± 0·02% respectively (P < 0·001). Irrespective of antigen disparity, the number of PKH‐labelled cells in the femur decreased by 30% and 50% after 1 and 3 d respectively (P < 0·001). Similar HE of naïve and irradiated cells suggested that the majority of cells (> 80%) were quiescent in the BM during the first 3 d. HE were twofold higher in busulphan‐myeloablated recipients (P < 0·001 vs non‐conditioned), and allogeneic transplantation resulted in 84 ± 9% donor chimaerism at 4 weeks. The HE of lin^– cells was 16‐fold higher than that of lin⁺ cells (P < 0·001), and the subset of lin^– SCA‐1⁺ cells was 4·6‐fold higher in the BM‐homed cell population (P < 0·001 vs lin^– cells). Approximately 1500 of the BM‐homed cells rescued 62–71% secondary syngeneic and allogeneic myeloablated recipients. Strikingly, the HE could be predicted during the first 3 d after transplantation by correcting the measurements performed in vivo for the enrichment of progenitors in donor inoculum, donor‐recipient antigen disparity and myeloablative conditioning.     

Rationale for bone marrow transplantation in the treatment of autoimmune diseases.

Transplantation of normal bone marrow from C3H/HeN nu/nu (H-2k) mice into young MRL/MP-lpr/lpr (MRL/l; H-2k) mice (less than 1.5 mo) prevented the development of autoimmune diseases and characteristic thymic abnormalities in the recipient mice. When female MRL/1 (greater than 2 mo) or male BXSB (H-2b) mice (9 mo) with autoimmune diseases and lymphadenopathy were lethally irradiated and then reconstituted with allogeneic bone marrow cells from young BALB/c nu/nu (H-2d) mice (less than 2 mo), the recipients survived for more than 3 mo after the bone marrow transplantation and showed no graft-versus-host reaction. Histopathological study revealed that lymphadenopathy disappeared and that all evidence of autoimmune disease either was prevented from developing or was completely corrected even after its development in such mice. All abnormal T-cell functions were restored to normal. The newly developed T cells were found to be tolerant of both bone marrow donor-type (BALB/c) and host-type (MRL/1 or BXSB) major histocompatibility complex (MHC) determinants. Therefore, T-cell dysfunction in autoimmune-prone mice can be associated with both the involutionary changes that occur in the thymus of the autoimmune-prone mice and also to abnormalities that reside in the stem cells. However, normal stem cells from BALB/c nu/nu donors can differentiate into normal functional T cells even in mice whose thymus had undergone considerable involution, as in the case of BXSB or MRL/1 mice in the present studies. These findings suggest that marrow transplantation may be a strategy ultimately to be considered as an approach to treatment of life-threatening autoimmune diseases in humans. T-cell dysfunction in autoimmune-prone mice previously attributed to involutionary changes that occur in the thymus of these mice may instead be attributed to abnormalities that basically reside in the stem cells of the autoimmune-prone mice.     

Effect of low-molecular-weight heparin on the commitment of bone marrow cells to liver sinusoidal endothelial cells in CCl(4)-induced liver injury.

BACKGROUND/AIMS: Recently liver regeneration by bone marrow transplantation has been proposed as an alternative source of functional liver cells. We investigate commitment of bone marrow cells (BMCs) to liver regeneration and the effect of dalteparin sodium (DS) on regeneration of the damaged liver caused by carbon tetrachloride (CCl(4)) administration in the mice. METHODS: Liver injury was produced in 8-week-old mice by treating with CCl(4) for 4 weeks. Thereafter, mice received a lethal dose of irradiation (10Gy) to whole body, followed by injection of 1x10(7) green fluorescent protein (GFP)-positive BMCs via the tail vein. DS (50IU/kg, intraperitoneally) was administered daily for 28 consecutive days starting at 1 day post-BMC transplantation. Lineage marker analysis of GFP-positive liver cells was performed immunostaining with a CD31 antibody. RESULT: Four weeks after BMC transplantation, GFP-positive cells in the CCl(4)-damaged liver could be detected in the lobule displaying a meshwork architecture extending from the periportal to pericentral regions, a pattern simulating sinusoidal lining. This localization of GFP-positive cells suggested that these cells were closely associated with sinusoidal endothelial cells. By staining the GFP-positive cells for CD31, it was confirmed that the majority of the GFP-positive cells are also positive for CD31. The GFP(+)CD31(+) cells were barely detected in the control group (1.0+/-1.2 per field). In marked contrast, a numerous number of GFP(+)CD31(+) cells were detected in the liver section obtained from the CCl(4)-induced liver damage group (3.8+/-1.3 per field, P<0.05 versus control). The number of GFP(+)CD31(+) cells in CCl(4) plus DS-treated group was further increased to 8.3+/-1.3 per field (P<0.05 versus CCl(4)-induced liver damage group). CONCLUSION: The majority of GFP-positive BMCs was committed to sinusoidal endothelial cells. DS promoted BMC differentiation into sinusoidal endothelial cells in the CCl(4)-damaged liver.     

Hematopoietic stem cells and endothelial cell precursors express Tie-2, CD31 and CD45

Early theories of tumor angiogenesis suggested that preexisting vessels surrounding the tumor were the principal source of the tumor vasculature but recent evidence suggests that endothelial progenitor cells (EPC) migrate from the marrow play an important role in developing the tumor blood supply. In a mouse model, in which the vascularization of a transplantable tumor was studied after bone marrow (BM) transplantation, we show that cells that express Tie-2, Sca-1, CD31 and CD45 function as both BM EPC and primitive hematopoietic stem cells. BM cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the endothelial lineage-specific Tie-2 promoter (Tie-2 /GFP) were used to reconstitute irradiated (12 Gy) wild-type mice. Five donor BM cell populations were studied: (1) whole BM; (2) Sca-1-enriched BMC; (3) GFP/Tie-2+, Sca-1+ BMC; (4) GFP/Tie-2-, Sca-1+ BMC and (5) Sca-1-depleted BMC. After 4 weeks, the mice were injected with Tg.AC tumor cells. Three weeks later, sections from the tumors were stained for CD31 and examined for Tie-2-driven GFP expression. BM-derived endothelial cells were found only in mice transplanted with bone marrow containing populations of Tie-2+, Sca-1+ cells. As few as 3500 of these cells were sufficient to radioprotect lethally irradiated mice. Thus, we conclude that a rare subset of BMC (approximately 4 x 10(-3)%) with the putative properties of hemangioblasts have an active Tie-2 promoter. Selection of Tie-2+Sca-1+ BMC enriches for marrow-derived EPCs that participate in tumor angiogenesis and cells that can provide hematopoietic reconstitution of marrow-ablated mice.     

Haematopoietic stem cells improve cardiac function after infarction without permanent cardiac engraftment

BACKGROUND: Transplantation of bone marrow derived adult stem cells (BMC) improves cardiac function after acute myocardial infarction (MI). However, the cell population mediating myocardial recovery and the fate of the transplanted cells are still controversial. AIMS: We determined the effects of Sca-1+ c-kit+ lin- haematopoietic BMC on cardiac function after MI and the cell fate after transplantation. METHODS: Sca-1+ c-kit+ lin- BMC of male donor C57BL/6 mice were transplanted by intravenous injection into syngenic females after permanent MI. LV dimensions and function were determined by echocardiography and cardiac magnetic resonance imaging, transplanted BMC were identified by Y chromosome DNA in situ hybridization. RESULTS: BMC treatment completely prevented LV dilation (LV end-diastolic volume BMC 70 +/- 16 microl vs. control 122 +/- 41 microl; p < 0.05) and improved fractional shortening (BMC 22.9 +/- 8% vs. control 15.4 +/- 8.4%; p < 0.05) and ejection fraction BMC 68.2 +/- 6.6% vs. control 52 +/- 14.3%; p < 0.05) as early as 3 days after transplantation, but did not decrease infarct size (BMC 27 +/- 6% vs. control 28 +/- 7%, p = n.s.). After 4 weeks, only sporadic cells of male origin were identified in infarcted hearts (< 0.01% of periinfarct cells). CONCLUSION: Intravenous injection of Sca-1+ c-kit+ lin- in BMC after MI improves LV dimensions and function without evidence for long term engraftment.     

Renin-angiotensin system expression in rat bone marrow haematopoietic and stromal cells

The existence of a bone marrow renin-angiotensin system (RAS) is evidenced by the association of renin, angiotensin converting enzyme (ACE), and angiotensin (Ang) II and its AT(1) and AT(2) receptors with both normal and disturbed haematopoiesis. The expression of RAS components by rat unfractionated bone marrow cells (BMC), haematopoietic-lineage BMC and cultured marrow stromal cells (MSC) was investigated to determine which specific cell types may contribute to a local bone marrow RAS. The mRNAs for angiotensinogen, renin, ACE, and AT(1a) and AT(2) receptors were present in BMC and in cultured MSC; ACE2 mRNA was detected only in BMC. Two-colour flow fluorocytometry analysis showed immunodetectable angiotensinogen, ACE, AT(1) and AT(2) receptors, and Ang II, as well as binding of Ang II to AT(1) and AT(2) receptors, in CD4(+), CD11b/c(+), CD45R(+) and CD90(+) BMC and cultured MSC; renin was found in all cell types with the exception of CD4(+) BMC. Furthermore, Ang II was detected by radioimmunoassay in MSC homogenates as well as conditioned culture medium. The presence of Ang II receptors in both haematopoietic-lineage BMC and MSC, and the de novo synthesis of Ang II by MSC suggest a potential autocrine-paracrine mechanism for local RAS-mediated regulation of haematopoiesis.     

Transplantation of bone marrow cells reduces CCl4‐induced liver fibrosis in mice

Background: We investigated the reversibility of liver fibrosis induced with a CCl₄ injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. Methods: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl₄: control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFP‐donor BM transplantation. Liver function tests and real‐time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl₄ for 2 days: granulocyte colony stimulating factor (G‐CSF) only, mononuclear cell (MNC) transplantation and Lin‐Sca‐1+c‐kit+haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellular matrix and liver fibrosis‐related factors. Results: The liver fibrosis induced by CCl₄ was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G‐CSF increased the recovery of the injured liver and the best results were seen in those mice administered the MNC fraction and Lin‐Sca‐1+c‐kit+HSC fraction, with no difference between the two groups. Conclusion: BMC transplantation and stem cell mobilization with G‐CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis.     

Transplantation of Autoimmune Potential. I. Development of Antinuclear Antibodies in H-2 Histocompatible Recipients of Bone Marrow from New Zealand Black Mice

Antinuclear autoantibodies (ANA) appeared in the plasma of lethally irradiated H-2d histocompatible DBA/2 and BALB/c mice several weeks after intravenous transplantation of 2 to 4 x 10(6) bone marrow cells from 3-week-old animals of the autoimmune New Zealand Black (NZB) strain. Little or no ANA development was observed in DBA/2 or BALB/c strains when syngeneic or nonautoimmune allogeneic marrow was grafted, or when NZB marrow was injected into untreated DBA mice or mice receiving 200 rads of x-irradiation. Transfer of 5 x 10(6) spleen cells from 8-day-old NZB mice into lethally irradiated BALB/c mice effected substantial ANA formation by the ninth day after transfer, compared to a 20-day latency following transfer of the same number of bone marrow cells. This earlier conversion with splenocytes may have been due to the presence of immunocompetent T and B cells, since stem cell numbers of the two tissues were similar. Transplantation of NZB marrow to lethally irradiated H-2 incompatible SJL/J (H-2s) and C57B1/6 (H-2b) strains brought about less ANA conversion than the transfer of compatible (SJL x NZB)F1 and (C57B1 x NZB)F1 marrow cells to the respective nonautoimmune SJL or C57B1 parental strain. Graft-versus-host reactions thus did not appear to play a requisite or determining role in the autoimmune development observed following grafting of NZB hemopoietic tissues. Reconstitution of lethally irradiated NZB mice with BALB/c or SJL/J bone marrow depressed the recurrence of ANA for 30 days, compared to rapid ANA recovery following NZB marrow injection. The characteristics that ultimately provoke or permit spontaneous auto-reactivity are inherent in the hemopoietic stem cell population of the NZB strain.