Conventional Bone Marrow Research Articles

Cord Blood Graft Engineering

Once considered solely medical waste and routinely discarded as part of the afterbirth, umbilical cord blood (CB) is now commonly used as a source of hematopoietic stem cells (HSCs) for patients in need of HSC transplantation (HSCT) (or, as more commonly referred to, bone marrow transplantation) for the treatment of both malignant and nonmalignant disorders. In fact, more than 20,000 CB transplantations (CBTs) have been performed since the first one in 1988 [1], and this number increases annually as more recent data demonstrate outcomes for CBT recipients paralleling those for conventional HSCT recipients. Numerous factors make CB a desirable source of HSCs, including ease of procurement and lack of donor attrition, with the ability to process and store the donor cells long term [2]. Importantly, CB donors can be used without the need for a “perfect” HLA match, thereby increasing donor access to HSCT, particularly for minority and mixed ethnicity patients, for whom a suitably matched related or unrelated donor may be difficult to locate. Moreover, despite the greater degree of HLA mismatch, CBT recipients are at decreased risk of developing chronic graft-versus-host disease (GVHD). The first decade of CBT experience was important in defining critical total nucleated cell (TNC) and CD34+ cell dose thresholds required for acceptable clinical outcomes, and in moving from related to unrelated donor CBT and from pediatric to adult patients. The limitations of this approach also were defined during this period, with low cell dose identified as the critical barrier. CBT recipients receive on average only 10% of the CD34+ stem/progenitor cells provided in conventional bone marrow grafts and only 5% of those provided in peripheral blood stem cell grafts, resulting in significant delays in engraftment and immune reconstitution, as well as an increased risk of graft failure and early transplantation-related mortality. Despite these limitations, the second decade of CBT was marked by improved outcomes, especially in adults, as better knowledge of cell dose requirements led to improved collection and the availability of units with higher cell doses. Better supportive care also contributed to these improved outcomes. Initial reports showed outcomes for CBT recipients on par with conventional HSCT recipients [3-5]. Finally, the advent of double CBT (dCBT) brought a significant reduction in the risk of graft failure, opening up the possibility of HSCT with CB for essentially all patients without a suitable donor [6]. Nonetheless, the use of dCBT did not produce faster neutrophil recovery or immune reconstitution, with cell dose remaining a major limitation. The advent of dCBT has, however, led to increased activity in the area of CB graft engineering, especially in the area of ex vivo expansion, with the double-unit platform allowing for manipulation of 1 unit and the ability to track both units in vivo. Ex vivo expansion of CB stem and progenitor cells to enhance engraftment is only one area of CB graft engineering currently under clinical investigation. As discussed below in more detail, the generation of immunotherapy from CB grafts is under active investigation both preclinically and now in the clinic as a means of overcoming the issues of delayed immune reconstitution in CBT recipients, with the generation of multivirus-specific cytotoxic T lymphocytes to treat viral infections. Relapse also remains a problem for all patients undergoing HSCT for hematologic malignancy. The ex vivo expansion of CB-derived T cells genetically modified to express CD19 or CD20 chimeric antigen receptors (CARs) to prevent relapse and the ex vivo generation of increased numbers of CB-derived NK cells as a means of better disease control are discussed below. This review focuses on these primary areas of graft engineering, but by no means represents an exhaustive list of the active research ongoing in the field of CB graft engineering. Other areas not covered in this review include the generation of regulatory T cells for GVHD prophylaxis, biological-based bioengineering approaches to enhance stem cell expansion, and preclinical research into the generation of CB-derived induced pluripotent stem cells. With continued advancement in the field, it is possible that a single CB unit could be manipulated based on an individual patient's greatest clinical need, with generation of specific cell types as clinically appropriate (Figure 1). Open in a separate window Figure 1 The potential of CB graft engineering.

Aging induced decline in T-lymphopoiesis is primarily dependent on status of progenitor niches in the bone marrow and thymus

Age-related decline in the generation of T cells is associated with two primary lymphoid organs, the bone marrow (BM) and thymus. Both organs contain lympho-hematopoietic progenitor/stem cells (LPCs) and non-hematopoietic stromal/niche cells. Murine model showed this decline is not due to reduced quantities of LPCs, nor autonomous defects in LPCs, but rather defects in their niche cells. However, this viewpoint is challenged by the fact that aged BM progenitors have a myeloid skew. By grafting young wild-type (WT) BM progenitors into aged IL-7R-/- hosts, which possess WT-equivalent niches although LPCs are defect, we demonstrated that these young BM progenitors also exhibited a myeloid skew. We, further, demonstrated that aged BM progenitors, recruited by a grafted fetal thymus in the in vivo microenvironment, were able to compete with their young counterparts, although the in vitro manipulated old BM cells were not able to do so in conventional BM transplantation. Both LPCs and their niche cells inevitably get old with increasing organismal age, but aging in niche cells occurred much earlier than in LPCs by an observation in thymic T-lymphopoiesis. Therefore, the aging induced decline in competence to generate T cells is primarily dependent on status of the progenitor niche cells in the BM and thymus.

Commentary on “Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning”

Transplantation of vascularized composite allografts (VCAs) could significantly affect the liver of the approximately 7 million Americans every year. These Americans would benefit from transplantation because they had undergone or need to undergo reconstructive surgery as the result of oncologic procedures, congenital anomalies, and trauma [ [1] Gander B. Brown C.S. Vasilic D. et al. Composite tissue allotransplantation of the hand and face: a new frontier in transplant and reconstructive surgery. Transplant Int. 2006; 19: 868 Crossref PubMed Scopus (77) Google Scholar ]. Just as important, it may also help bring us a step closer to the “Holy Grail” of transplantation, which is the induction of donor-specific immunologic tolerance. The reason is because, to achieve a state of tolerance, the most promising approach appears to be the creation of mixed hematopoietic chimerism [ [2] Bradley J.A. Induction of transplant tolerance through mixed hematopoietic chimerism. Am J Transplant. 2012; 12: 1073 Crossref PubMed Scopus (3) Google Scholar ]. The significance of VCA transplantation lies in the fact that by definition, VCA already contain hematopoietic tissue, which can lead to chimerism with reduced conditioning. Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioningJournal of Surgical ResearchVol. 178Issue 2PreviewCotreatment with regulatory T cells (Treg) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether Treg treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. Full-Text PDF

Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning

BackgroundCotreatment with regulatory T cells (Treg) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether Treg treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. MethodsRecipient Lewis rats treated (day 0) with or without naturally sorted Treg (3 × 106) from Lewis rat spleen and lymph nodes received costimulation blockade (anti-CD154 monoclonal antibody, days 0 and 1 and CTLA-4 immunoglobin, days 2, 4, and 6), rapamycin (days −1, 0, and 2), and concurrent transplantation of fully mismatched allogeneic donor VCAs (day 0) from the Brown Norway rat hindlimb containing VBMT. The mixed chimerism level was assessed monthly using flow cytometry. Survival of VCAs and occurrence of graft-versus-host disease were assessed clinically and histologically. ResultsThe combination of Treg and VBMT treatment led to long-term multilineage hematopoietic mixed chimerism (12–18%) and long-term donor-specific tolerance to VCAs (89% acceptance rate). Neither stable mixed chimerism nor VCA acceptance was observed in recipients without Treg treatment. Graft-versus-host disease did not occur in the VBMT recipients. ConclusionsCotreatment with Treg and VBMT created stable mixed chimerism and induced long-term donor-specific tolerance to VCAs without requiring cytoreductive conditioning. This noncytoreductive Treg-VBMT protocol has potential for clinical application in VCAs.

Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis

Mesenchymal stem cells (MSCs) hold promise for cell-based therapy in regenerative medicine. To date, MSCs have been obtained from conventional bone marrow via a highly invasive procedure. Therefore, MSCs are now also isolated from sources such as adipose tissue, cord blood and cord stroma, a subject of growing interest. As the characterization and differentiation potential of adipose-derived MSCs (AD-MSCs) and bone-marrow-derived MSCs (BM-MSCs) have not been documented, we have evaluated and compared the characteristics of both MSC types by qualitative and quantitative analyses. Both cell types show similar morphology and surface protein expression, being positive for stromal-associated markers and negative for hematopoietic and endothelial markers. The colony-forming potential of AD-MSCs is distinctly higher than that of BM-MSCs. Nonetheless, similar adipogenic and osteogenic differentiation is observed in both groups of MSCs. Cytochemical qualitative analysis and calcium mineralization demonstrate higher levels toward osteogenic differentiation in BM-MSCs than in AD-MSCs. On the contrary, the percentage of Nile red oil staining for differentiated adipocytes is higher in AD-MSCs than in BM-MSCs. Quantitative real-time polymerase chain reaction shows similar patterns of osteogenic- and adipogenic-associated gene expression in both cell types. Each of theMSCs respond in functional analysis by exhibiting unique properties at the differentiation level according to their micro-environmental niche. Thus, quantitative analysis might be a valuable means of describing stem cell multipotency, in addition to qualitative investigation.

Vascularized Bone Marrow Transplantation Model in Rats as an Alternative to Conventional Cellular Bone Marrow Transplantation: Preliminary Results

The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMT) versus conventional bone marrow transplantation (BMT). In one group, a VBMT model consisted of donor Brown Norway rat hind limb heterotopic transplanted on recipient Lewis rats. An intravenous infusion of donor bone marrow cells in suspension equivalent to that grafted in the vascularized femur limb was administered intravenously to recipient rats in the second group. Cellular microchimerism was investigated in recipients of VBMT versus BMT. Donor-derived cells could be detected in VBMT recipients at 30 and 60 days but not in recipients of intravenous suspension of BMC. VBMT provides a theoretical alternative to conventional cellular bone marrow transplantation by addressing crucial clinical problems such as failure of engraftment or graft-versus-host disease.

Influence of Coping Style on Symptom Interference Among Adult Recipients of Hematopoietic Stem Cell Transplantation

To investigate the influence of coping style on interference caused by a variety of common post-treatment symptoms after hematopoietic stem cell transplantation. Longitudinal; secondary analysis of data from the original study that examined health-related quality-of-life variables (e.g., depression, well-being) in adult patients treated with conventional bone marrow transplantation or depleted T-cell bone marrow transplantation. Fifteen university medical centers in the United States. 105 adult recipients of hematopoietic stem cell transplantation. Patients were assessed via telephone-based interviews for coping style at baseline and for symptom interference in daily living six months post-treatment. Coping style and symptom interference. Neither age nor gender predicted symptom interference, with the exception of chronic graft-versus-host disease, where older patients experienced more interference at six months, and breathing symptoms, for which women experienced more interference than men at six months. Avoidant coping style at baseline predicted increased interference from symptoms, but emotion-focused and instrumental coping styles did not predict decreased interference. A generalized avoidant coping style before treatment increased interference from common cancer symptoms six months after hematopoietic stem cell transplantation. An intervention to teach alternate coping strategies should be implemented prior to treatment and tested for prevention of symptom-related life interference.

Bid is a positive regulator for donor-derived lymphoid cell regeneration in γ-irradiated recipients

Hematopoietic regeneration is regulated by cell survival proteins, such as the Bcl-2 family. Bid, a BH3-only protein of the Bcl-2 family, has multiple cellular functions and is involved in a variety of physiological or pathological conditions. We attempted to define its role in hematopoietic cell repopulation under the stress condition of bone marrow transplantation. We performed conventional or competitive bone marrow transplantation with donor hematopoietic cells from Bid(-/-) or Bid(+/+) mice. Flow cytometry was used for quantification of hematopoietic stem cells, hematopoietic progenitor cells, and differentiated cells in different lineages (T, B, and myeloid cells). Single cell culture and homing assays were performed to further evaluate hematopoietic stem cell functions. Hematopoietic progenitor cells were also measured by the colony-forming cell culture. Contrary to the widely recognized role of Bid as a pro-apoptotic protein, the absence of Bid significantly reduced the reconstitution of donor hematopoietic cells in γ-irradiated recipients. Interestingly, however, numbers of hematopoietic stem cells and hematopoietic progenitor cells and their functions were not overtly altered. Instead, the regeneration of donor T and B cells was significantly impaired in the absence of Bid. Further analysis indicated an accumulation of the triple-negative T-cell population in the thymus, and pro-B cells in the bone marrow. Our current study demonstrates a positive impact of Bid on hematopoietic regeneration mainly due to its unique effects on donor lymphopoiesis in the transplant recipients.

Treatment of Early Avascular Necrosis of Femoral Head by Small Intestinal Submucosal Matrix With Peripheral Blood Stem Cells

Background Avascular necrosis of the femoral head (ANFH) is a highly mutilating disease. There are no effective ways to treat early femoral head ischemia. Peripheral blood stem cell (PBSC) transplantation may be superior to conventional bone marrow transplantation. Small intestinal submucosae matrix (SIS) is composed of highly conserved collagens, glycoproteins, proteoglycans, and glycoaminoglycans in their natural configuration and concentrations. When implanted in a number of microenvironments in vivo, SIS has been used to induce proliferation, remodeling, and regeneration of host tissues. This study was designed to verify the curative effects of PBSC and SIS transplantation–induced vascular regeneration to improve ischemic femoral head necrosis in rabbits. Methods 32 New Zealand white rabbits underwent ischemic femoral head necrosis modeling in both hindlimbs by liquid-nitrogen refrigeration. All rabbits were intraperitoneally injected with grannlocytectomy–stimulating factor (250 μg/kg/d), except for normal control subjects injected with equivalent saline solution. After separation of peripheral blood stem cells (PBSCs), 64 femoral heads in 32 rabbits were randomly divided into 4 groups: group A, cancellous bone and peripheral blood stem cells cultured with small intestinal submucosa; group B, cancellous bone and PBSCs; group C, cancellous bone autografts; and group D, no treatment. The specimens were harvested at 4 and 8 weeks after surgery. All specimens were examined to observe angiogenesis and osteogenesis repairing the avascular necrosis of the femoral head by using gross observation, x-ray, histology, and immunohistochemical staining. Results In 4 weeks after peripheral stem cell transplantation, the standing ability and activity of the transplanted hindlimbs were improved remarkably, but there were no obvious changes in the control limbs. X-rays showed a greater density of grafts than the host bone in groups A,B, and group C was unchanged at 4 weeks. Histology revealed many osteoprogenitor cells and osteoblasts and no inflammatory cell infiltration at 4 weeks with much new bone formed at 8 weeks in group A and at 4 weeks in group B. The cancellous bone autograft was absorbed completely at 8 weeks in group C. There was little osteoid tissue formed in group D at 8 weeks. The zone of new bone formation in group A was greater than that in group B ( P < .05), but there was no significant difference between groups A and C ( P > .05). Immunohistochemical staining with CD31 mouse antibody showed greater amounts and zones of new blood vessels in groups A and B at 4 and 8 weeks and little evidence in group D. There was no significant difference between groups A and B ( P > .05) and significant differences between groups A and B versus C and D ( P < .05). Conclusion Transplantation of PBSCs cultured with SIS effectively improved ischemic femoral head necrosis.

Artificial Niche Combining Elastomeric Substrate and Platelets Guides Vascular Differentiation of Bone Marrow Mononuclear Cells

Bone marrow-derived progenitor cells are promising cell sources for vascular tissue engineering. However, conventional bone marrow mesenchymal stem cell expansion and induction strategies require plating on tissue culture plastic, a stiff substrate that may itself influence cell differentiation. Direct scaffold seeding avoids plating on plastic; to the best of our knowledge, there is no report of any scaffold that induces the differentiation of bone marrow mononuclear cells (BMNCs) to vascular cells in vitro. In this study, we hypothesize that an elastomeric scaffold with adsorbed plasma proteins and platelets will induce differentiation of BMNCs to vascular cells and promote vascular tissue formation by combining soft tissue mechanical properties with platelet-mediated tissue repairing signals. To test our hypothesis, we directly seeded rat primary BMNCs in four types of scaffolds: poly(lactide-co-glycolide), elastomeric poly(glycerol sebacate) (PGS), platelet-poor plasma-coated PGS, and PGS coated by plasma supplemented with platelets. After 21 days of culture, osteochondral differentiation of cells in poly(lactide-co-glycolide) was detected, but most of the adhered cells on the surface of all PGS scaffolds expressed calponin-I and α-smooth muscle actin, suggesting smooth muscle differentiation. Cells in PGS scaffolds also produced significant amount of collagen and elastin. Further, plasma coating improves seeding efficiency, and platelet increases proliferation, the number of differentiated cells, and extracellular matrix content. Thus, the artificial niche composed of platelets, plasma, and PGS is promising for artery tissue engineering using BMNCs.

Absence of Graft Failure and Excellent Long Term Survival Following Fludarabine-Based Reduced Intensity Hematopoietic Stem Cell Transplantation In Heavily Transfused Patients with ATG-Refractory Severe Aplastic Anemia and Other Bone Marrow Failure Syndromes

AbstractAbstract 517Heavily transfused and alloimmunized patients with bone marrow failure syndromes including aplastic anemia have an increased risk of graft rejection following conventional allogeneic bone marrow transplantation. Results from pilot trials suggest the addition of fludarabine to the conditioning regimen reduces the risk of graft rejection in patients at high risk for this complication. Here we report the results of a fludarabine-based transplant approach in 56 patients with severe aplastic anemia (SAA) or other bone marrow failure syndromes (SAA n=31, MDS-RA n=6, PNH n=16, PRCA n=2, DBA n=1) who were transplanted from May 1999 to November 2008 at the NHLBI. Forty one percent of patients were found to be alloimmunized (median 82% PRA) prior to transplantation as a consequence of prior transfusions. Seventy three percent of patients had received antibody-based immunesuppressive therapy at a median of 303 days (range 21 to 2588) prior to transplantation (horse-ATG n=34, rabbit-ATG n=5, alemtuzumab n=1 and daclizumab n=1). Conditioning with fludarabine (25 mg/m2 × 5 days), ATG (40mg/kg × 4 days) and cyclophosphamide (60mg/kg × 2 days) was followed by infusion of an un-manipulated G-CSF mobilized allograft from an HLA matched (n=52) or single antigen mismatched (n=4) relative. GVHD prophylaxis consisted of cyclosporine (CSA) either alone (n=2) or combined with mycophenolate mofetil (n=10) or mini-dose methotrexate (n=44). The median CD34+ cell dose was 6.6 × 106 cells/kg (range 1.7 to 21.1 × 106 cells/kg) and the median CD3+ cell dose was 2.6 × 108 cells/kg (range 0.5 to 6.9 x108 cells/kg). Nearly half (46 %) of patients received an ABO incompatible allograft (major mismatch n=15; minor mismatch n=11). Despite a high prevalence of pre-transplant alloimmunization, graft rejection and/or graft failure did not occur, with all patients achieving sustained donor engraftment in both myeloid and T-cell lineages. The median time to achievement of full donor (>= 95%) myeloid and T-cell chimerism was 15 and 30 days respectively. Neutrophil and platelet recovery occurred at a median 15 (range 6 to 24) and 12 (range 5 to 168) days respectively. Major ABO incompatibility was associated with delayed donor erythropoiesis; reticulocyte recovery (> 60 K/μ L on two occasions) occurred at a median 17 days in those without major ABO incompatibility and 42 days in the recipients of a major ABO mismatched graft, where clearance anti-donor isohemagglutinins was delayed a median 171 days following transplantation. CMV reactivation occurred in 31/50 (62%) patients at risk although no patients died from CMV related mortality. With a median follow-up of 4.5 years (range 1.8–11 years) in surviving patients, overall survival was 87.1%. There were 5 treatment related deaths with two attributable to steroid refractory acute GVHD and one attributable to extensive chronic GVHD. The cumulative incidence of Grade II-IV, III-IV and steroid refractory acute GVHD was 51.8%, 30.4% and 21.4% respectively. The cumulative incidence of chronic GVHD was 72% (23.2% limited and 48.9% extensive), with 42.5% who developed cGVHD having resolution of symptoms allowing discontinuation of systemic immunosuppressive therapy. Conclusion: Fludarabine-based allogeneic peripheral blood stem cell transplantation achieves excellent donor engraftment and long-term disease free survival in heavily transfused and alloimmunized patients with ATG refractory SAA and other nonmalignant hematological disorders associated with bone marrow failure. Efforts to reduce the high incidence of GVHD associated with this approach without increasing the risk of graft failure by manipulating the cellular content of the allograft are currently being explored. Disclosures:No relevant conflicts of interest to declare.

Remitting&amp;ndash;relapsing multiple sclerosis patient refractory to conventional treatments and bone marrow transplantation who responded to natalizumab

Bone marrow transplantation (BMT) was introduced as a treatment option 15 years ago for severe, drug-resistant multiple sclerosis (MS). Up until now, BMT has been undertaken in relatively few patients worldwide, with moderate success, and recent studies suggest that patients with early, highly aggressive MS benefit most from this treatment. In this work, we determined peripheral blood lymphocyte populations in a patient (patient A) with remitting–relapsing multiple sclerosis (RR-MS), refractory to conventional treatments, and who underwent BMT, relapsed, and has been treated with natalizumab for the last 22 months. Eleven other RR-MS patients in the acute phase of the disease, untreated or treated with interferon-beta, and 20 healthy subjects served as controls. Natalizumab treatment in patient A resulted in lymphocytosis and increased levels of CD20+/CD20+CD5+ B cells and T regulatory cells (Tregs). The patient maintained relatively low levels of T cells, T helper cells, memory T helper cells, and naive cytotoxic T cells, and very low levels of naive T helper cells and natural killer cells throughout. The Tregs of patient A post-treatment with natalizumab responded well in culture to a peptide mapping to a myelin basic protein antigenic epitope (mean 42% increase) compared with Tregs of healthy controls (mean 15% increase) whereas Tregs of the RR-MS controls or patient A prenatalizumab treatment either did not respond or responded adversely to the peptide (mean 3% and 21% decreases, respectively). Since the beginning of natalizumab treatment, patient A has had no relapses, and his Expanded Disability Status Score has improved. From the parameters studied, Treg responsiveness to autoantigens seems to be an important differentiating factor in RR-MS progression.

Granulocyte-Colony Stimulating Factor Reactivates Human Cytomegalovirus in a Latently Infected Humanized Mouse Model

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in organ transplant recipients. The use of granulocyte-colony stimulating factor (G-CSF)-mobilized stem cells from HCMV seropositive donors is suggested to double the risk of late-onset HCMV disease and chronic graft-versus-host disease in recipients when compared to conventional bone marrow transplantation with HCMV seropositive donors, although the etiology of the increased risk is unknown. To understand mechanisms of HCMV transmission in patients receiving G-CSF-mobilized blood products, we generated a NOD-scid IL2Rγ(c)(null)-humanized mouse model in which HCMV establishes latent infection in human hematopoietic cells. In this model, G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. In addition to establishing a humanized mouse model for systemic and latent HCMV infection, these results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients.

Transplantation tolerance through mixed chimerism

The major factors that limit the success of organ transplantation are the host immune response to the foreign graft and the adverse effects of the chronic immunosuppressive therapy required to suppress this immune response. Deliberately establishing tolerance towards the donor tissue by reprogramming the immune system of the recipient thus holds great promise in improving organ transplant survival and eliminating the untoward effects of chronic drug therapy. The transplantation of donor bone marrow into recipients who are appropriately conditioned to allow development of either full or mixed chimerism has long been recognized to effectively induce donor-specific tolerance. Despite the demonstrated effectiveness of this technique, use of the mixed chimerism strategy in regular clinical practice has been hampered by the toxic side effects inherent to conventional bone marrow transplantation protocols. This Review addresses recent advances in preclinical and clinical studies inducing transplantation tolerance through mixed chimerism and discusses both the potential and the challenges of this approach.

Living related liver transplant following bone marrow transplantation from same donor: Long-term survival without immunosuppression

We report long-term (seven yr) immunological tolerance in a 16-yr-old boy, to a liver allograft donated by his father following a bone marrow transplant at age 2.5 yr from the same donor. The bone marrow transplant was complicated by severe GVHD leading to liver failure and the ensuing need for a liver transplant, performed under planned avoidance of immunosuppression. At one wk post-transplant, although a liver biopsy was histologically compatible with acute rejection, favorable clinical and biochemical evolution precluded initiating immunosuppressive therapy, thus highlighting the need for caution when interpreting early histological changes so that administration of unnecessary immunosuppression can be avoided. Induction of tolerance in transplant recipients remains an elusive goal. In those patients who had received conventional bone marrow transplants and had endured the consequences of GVHD, development of macrochimerism may allow immunosuppression-free solid organ transplantation from the same donor.

Adipose Tissue-Derived Mesenchymal Stem Cells Facilitate Hematopoiesis in Vitro and in Vivo: Advantages Over Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.

Detection of Bone Marrow Metastases of Neuroblastoma With Immunohistochemical Staining of CD56, Chromogranin A, and Synaptophysin

Neuroblastoma is one of the most common solid tumors occurring in childhood. Bone marrow evaluation is an important part of clinical staging in neuroblastoma patients. In view of the difficulty of detecting neuroblastoma cells with conventional bone marrow aspirate smears and biopsy (BMB) and specimens in cases in which metastasis is not prominent, we propose the use of immunohistochemistry (IHC) as a potential diagnostic tool. We examined 116 BMB and 115 bone marrow clot (BMC) specimens from 60 newly diagnosed neuroblastoma patients for tumor cells. Neuroendocrine IHC markers, such as CD56, chromogranin A, and synaptophysin were applied to diagnose neuroblastoma. Eighteen out of the 60 patients (25.4%) displayed BM metastasis, as observed with conventional hematoxylin and eosin staining. IHC staining of BMC sections was generally more sensitive than that of BMB sections for tumor cell detection. We detected tumors in 5 and 7 additional hematoxylin and eosin-negative BMB and BMC sections, respectively, using CD56. Overall, CD56 or a combination of CD56 and chromogranin A was effective in detecting neuroblastoma cells. IHC analysis of BMB and BMC sections is warranted as a routine component of the diagnostic work-up of neuroblastoma to overcome discrepancies between routine smears and IHC stains.

Bone Marrow Cellularity Measurement by Myelocrit

The bone marrow (BM) cellularity has been used as a major index for the evaluation of hematologic diseases and malignancies. However, the microscopic evaluation lacks objectivity because of considerable variations among different observers. We measured myelocrit cellularity as an objective method to evaluate the cellularity. Between November 2007 and January 2008, 489 consecutive BM aspirates including 25 cases of AML D7 marrow (7 days after initiation of chemotherapy) were examined. The conventional BM cellularity was evaluated using BM needle biopsies after hematoxylin-eosin stain. EDTA-anticoagulated BM aspirates were put into the Wintrobe tubes, centrifuged and the thickness of 5 layers from the bottom was measured: RBC, buffy coat (BC), plasma, BM particle, and fat layers. The myelocrit cellularity was defined as the ratio of BC to the BC plus fat layers. Both of the thickness of BC layer (r=0.721, P=0.000) and the myelocrit cellularity (r=0.735, P=0.000) correlated well with the conventional cellularity. However, the AML D7 BM cellularity correlated with BC layer (r=0.589, P=0.002), but not with the myelocrit cellularity (r=0.281, P=0.231). The cellularity of the BM other than AML D7 marrow showed a better correlation with the myelocrit cellularity (r=0.826, P=0.000) than the BC layer (r=0.713, P=0.000). The myelocrit is a simple, reproducible and objective method to determine the BM cellularity. For accurate assessment of BM cellularity, measurement of the thickness of BC layer in AML D7 BM and of the myelocrit cellularity in other BM samples has better be used.

Mobilization of Hematopoietic Stem Cells in a Thalassemic Mouse Model: Implications for Human Gene Therapy of Thalassemia

Granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cells may become the preferable source of hematopoietic stem cells (HSCs) for gene therapy because of the higher yield of cells compared with conventional bone marrow harvesting. A G-CSF-associated risk of splenic rupture has been recognized in normal donors of HSCs, but limited information is available about the G-CSF effect in the presence of splenomegaly and extramedullary hematopoiesis. We investigated the G-CSF effect in a thalassemic mouse model (HBB(th-3)) as compared with a normal strain (C57BL/6), in terms of safety, mobilization efficacy, and distribution of stem cells among hematopoietic compartments. There was no death or clinical sequelae of splenic rupture in G-CSF-treated animals of either strain; however, hemorrhagic infarcts in the spleen were detected with low frequency in G-CSF-treated HBB(th-3) mice (12.5%). HBB(th-3) mice mobilized less effectively than C57BL/6 mice (Lin(-)Sca-1(+)c-Kit(+) cells/microl of peripheral blood mononuclear cells [PBMCs]: 90 +/- 55 vs. 255 +/- 174, respectively, p = 0.01; CFU-GM/ml PBMCs: 390 +/- 262 vs. 1131 +/- 875, p = 0.01) because of increased splenic trapping of hematopoietic stem and progenitor cells (Lin(-)Sca-1(+)c-Kit(+) cells per spleen (x10(5)): 487 +/- 35 vs. 109 +/- 19.6, p = 0.01; CFU-GM per spleen (x10(2)): 1470 +/- 347 vs. 530 +/- 425, p = 0.0006). Splenectomy restored the mobilization proficiency of thalassemic mice at comparable levels to normal mice and resulted in the development of a hematopoietic compensatory mechanism in the thalassemic liver that protected splenectomized mice from severe anemia. Our data imply that, in view of human gene therapy for thalassemia, either multiple cycles or alternative ways of mobilization may be required for a sufficient yield of transplantable HSCs. In addition, strategies to minimize the risk of G-CSF-induced splenic infarcts should be explored in a clinical setting.

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry, PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis.