The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment.
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ABSTRACT:
SUBMITTER: Wernig G
PROVIDER: S-EPMC1187970 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mesenchymal stem cell (MSC) therapy is a promising approach to curing bone diseases and disorders. In treating genetic bone disorders, MSC therapy is local or systemic transplantation of isolated and in vitro proliferated MSC rather than bone marrow transplantation. Recent evidence showed that bone marrow MSC engraftment to bone regeneration has been controversial in animal and human studies. Here, our modified bone marrow transplantation (BMT) method solved this problem. Like routine BMT, our modified method involves three steps: (i) isolation of bone marrow cells from the donor, (ii) whole-body lethal irradiation to the recipient, and (iii) injection of isolated bone marrow cells into irradiated recipient mice via the tail vein. The significant modification is imported at the bone marrow isolation step. While the bone marrow cells are flushed out from the bone marrow with the medium in routine BMT, we applied the enzymes' (collagenase type 4 and dispase) integrated medium to wash out the bone marrow cells. Then, cells were incubated in enzyme integrated solution at 37°C for 10 minutes. This modification designated BMT as collagenase-integrated BMT (c-BMT). Notably, successful engraftment of bone marrow MSC to the new bone formation, such as osteoblasts and chondrocytes, occurs in c-BMT mice, whereas routine BMT mice do not recruit bone marrow MSC. Indeed, flow cytometry data showed that c-BMT includes a higher proportion of LepR+, CD51+, or RUNX2+ non-hematopoietic cells than BMT. These findings suggested that c-BMT is a time-efficient and more reliable technique that ensures the disaggregation and collection of bone marrow stem cells and engraftment of bone marrow MSC to the recipient. Hence, we proposed that c-BMT might be a promising approach to curing genetic bone disorders. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Project description:Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.
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Project description:Duchenne muscular dystrophy (DMD) is a severe muscular disorder caused by mutations in the dystrophin gene. It leads to respiratory and cardiac failure and premature death at a young age. Although recent studies have greatly deepened the understanding of the primary and secondary pathogenetic mechanisms of DMD, an effective treatment remains elusive. In recent decades, stem cells have emerged as a novel therapeutic product for a variety of diseases. In this study, we investigated nonmyeloablative bone marrow cell (BMC) transplantation as a method of cell therapy for DMD in an mdx mouse model. By using BMC transplantation from GFP-positive mice, we confirmed that BMCs participate in the muscle restoration of mdx mice. We analyzed both syngeneic and allogeneic BMC transplantation under different conditions. Our data indicated that 3 Gy X-ray irradiation with subsequent BMC transplantation improved dystrophin synthesis and the structure of striated muscle fibers (SMFs) in mdx mice as well as decreasing the death rate of SMFs. In addition, we observed the normalization of neuromuscular junctions (NMJs) in mdx mice after nonmyeloablative BMC transplantation. In conclusion, we demonstrated that nonmyeloablative BMC transplantation could be considered a method for DMD treatment.
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Project description:In vitro amplification of polymorphic genetic markers, especially short tandem repeats (STRs), has become standard laboratory practice in the monitoring of allogeneic bone marrow transplant patients. After initial analysis of donor and recipient samples at multiple loci before transplantation, one or more loci are used to follow engraftment status in subsequent specimens. We describe an unusual pattern of STRs in a transplanted patient with a prior history of refractory acute myelogenous leukemia. DNA chimerism studies showed a lack of engraftment at 1 and 2 months after transplantation. Atypical minor peaks occurred at each of three STR loci in the pre-transplant and 2-month post-transplant recipient samples. However, these peaks were of equal amplitude as the major corresponding allele in the 1-month post-transplant sample. A history of myelodysplasia with specific chromosomal deletions before the patient's acute myelogenous leukemia diagnosis appears to explain the spurious peaks. STR analysis of blood and archival paraffin-embedded tissues collected from the patient at various time points before transplantation reflected the evolution, progression, and response to therapy of the myelodysplasia. The case illustrates the need for comprehensive evaluation of pertinent clinical and laboratory data during engraftment monitoring to identify potential sources for error in interpretation of STR analysis.
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Project description:Suicide gene therapy using gene-directed enzyme prodrug therapy (GDEPT) is based on delivering a gene-encoded enzyme to cells that converts a nontoxic prodrug into its toxic metabolite. The bystander effect is thought to compensate for inefficiencies in delivery and expression because the produced toxic metabolite can spread to adjacent non-expressing cells.The purpose of this study was to assess the significance of bystander effect in GDEPT over the long term in vivo.We performed experiments using mixtures of yeast cytosine deaminase (yCD) expressing and empty vector (EV) containing cells. First, the bystander effect was assessed in various ratios of colon cancer cell lines RKO with yCD/EV in 2D and 3D culture. Next, tumors raised from RKO with yCD/EV in mice were treated with the prodrug 5-fluorocytosine (5-FC) for 42 days to assess bystander effect in vivo. Cell types constituting relapsed tumors were determined by 5-FC treatment and PCR.We were able to demonstrate bystander effect in both 2D and 3D. In mice, tumors initially regressed, but they all eventually recurred including those produced from 80% yCD expressing cells. Cells explanted from the recurrent tumors demonstrated that suicide gene expressing cells had been selected against during in vivo treatment with 5-FC.We conclude that gene therapy of malignant tumors in patients using the yCD/5-FC system will require targeting well over 80% of the malignant cells, and therefore will likely require improved bystander effect or repeated treatment.
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Project description:Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.
| S-EPMC4827391 | biostudies-literature
Project description:The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells.
| S-EPMC2570168 | biostudies-literature
Project description:BackgroundAdult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells.MethodsFemale mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations.ResultsThe frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0-1.6% with whole marrow and 0.6-1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any cytokeratin mRNA in SP or bone marrow samples by RT-PCR.ConclusionThe appearance of bone marrow derived cells in the tracheal epithelium is enriched by detergent-induced tissue damage and the majority of these cells express an epithelial marker. The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo.
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Project description:Despite estimates that, each year, as many as 300 million dengue virus (DENV) infections result in either no perceptible symptoms (asymptomatic) or symptoms that are sufficiently mild to go undetected by surveillance systems (inapparent), it has been assumed that these infections contribute little to onward transmission. However, recent blood-feeding experiments with Aedes aegypti mosquitoes showed that people with asymptomatic and pre-symptomatic DENV infections are capable of infecting mosquitoes. To place those findings into context, we used models of within-host viral dynamics and human demographic projections to (1) quantify the net infectiousness of individuals across the spectrum of DENV infection severity and (2) estimate the fraction of transmission attributable to people with different severities of disease. Our results indicate that net infectiousness of people with asymptomatic infections is 80% (median) that of people with apparent or inapparent symptomatic infections (95% credible interval (CI): 0-146%). Due to their numerical prominence in the infectious reservoir, clinically inapparent infections in total could account for 84% (CI: 82-86%) of DENV transmission. Of infections that ultimately result in any level of symptoms, we estimate that 24% (95% CI: 0-79%) of onward transmission results from mosquitoes biting individuals during the pre-symptomatic phase of their infection. Only 1% (95% CI: 0.8-1.1%) of DENV transmission is attributable to people with clinically detected infections after they have developed symptoms. These findings emphasize the need to (1) reorient current practices for outbreak response to adoption of pre-emptive strategies that account for contributions of undetected infections and (2) apply methodologies that account for undetected infections in surveillance programs, when assessing intervention impact, and when modeling mosquito-borne virus transmission.
| S-EPMC5933708 | biostudies-literature
Project description:Lack of expression of dystrophin leads to degeneration of muscle fibers and infiltration of connective and adipose tissue. Cell transplantation therapy has been proposed as a treatment for intractable muscle degenerative disorders. Several reports have demonstrated the ability of bone-marrow derived cells (BMDC) to contribute to non-haematopoietic tissues including epithelium, heart, liver, skeletal muscle and brain following transplantation by means of fusion and reprogramming. A key issue is the extent to which fusion and reprogramming can occur in vivo, particularly under conditions of myogenic deterioration.To investigate the therapeutic potential of bone marrow transplantation in monogenetic myopathy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into non-irradiated c-kit receptor-deficient (W?¹) mdx mice. This model allows BMDC reconstitution in the absence of irradiation induced myeloablation. We provide the first report of BMDC fusion in a W?¹Dmd(mdx) deficient mouse model.In the absence of irradiation induced injury, few GFP+ cardiomyocytes and muscle fibres were detected 24 weeks post BMT. It was expected that the frequency of fusion in the hearts of W?¹Dmd(mdx) mice would be similar to frequencies observed in infarcted mice. Although, it is clear from this study that individual cardiomyocytes with monogenetic deficiencies can be rescued by fusion, it is as clear that in the absence of irradiation, the formation of stable and reprogrammed fusion hybrids occurs, with the current techniques, at very low levels in non-irradiated recipients.
| S-EPMC3225365 | biostudies-literature