Intra-bone Marrow Injection Research Articles

Interleukin-12 sustained release system promotes hematopoietic recovery after radiation injury.

The continuous production of mature blood cell lineages is maintained by hematopoietic stem cells but they are highly susceptible to damage by ionizing radiation (IR) that induces death. Thus, devising therapeutic strategies that can mitigate hematopoietic toxicity caused by IR would benefit acute radiation syndrome (ARS) victims and patients receiving radiotherapy. Herein, we describe the preparation of an injectable hydrogel formulation based on Arg-Gly-Asp-alginate (RGD-Alg) and Laponite using a simple mixing method that ensured a slow and sustained release of interleukin-12 (IL-12) (RGD-Alg/Laponite@IL-12). The local administration of RGD-Alg/Laponite@IL-12 increased survival rates and promoted the hematopoietic recovery of mice who had received sublethal-dose irradiation. Local intra-bone marrow (intra-BM) injection of RGD-Alg/Laponite@IL-12 hydrogel effectively stimulated IL12 receptor-phosphoinositide 3-kinase/protein kinase B (IL-12R-PI3K/AKT) signaling axis, which promoted proliferation and hematopoietic growth factors secretion of BM mesenchymal stem/stromal cells. This signaling axis facilitates the repair of the hematopoietic microenvironment and plays a pivotal role in hematopoietic reconstitution. In conclusion, we describe a biomaterial-sustained release of IL-12 for the treatment of irradiated hematopoietic injury and provide a new therapeutic strategy for hematopoietic ARS.

Intra-bone marrow injection with engineered Lactococcus lactis for the treatment of metastatic tumors: Primary report

Bone marrow has the capacity to produce different types of immune cells, such as natural killer cells, macrophages, dendritic cells (DCs) and T cells. Improving the activation of immune cells in the bone marrow can enhance the therapy of bone metastases. Previously, we designed an engineered probiotic Lactococcus lactis, capable of expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand (FOLactis), and proved that it can induce the activation and differentiation of several immune cells. In this research, we successfully establish mouse models of bone metastasis, lung metastasis and intraperitoneal dissemination, and we are the first to directly inject the probiotics into the bone marrow to inhibit tumor growth. We observe that injecting FOLactis into the bone marrow of mice can better regulate the immune microenvironment of tumor-bearing mice, resulting in a tumor-suppressive effect. Compared to subcutaneous (s.c.) injection, intra-bone marrow (IBM) injection is more effective in increasing mature DCs and CD8+ T cells and prolonging the survival of tumor-bearing mice. Our results confirm that IBM injection of FOLactis reprograms the immune microenvironment of bone marrow and has remarkable effectiveness in various metastatic tumor models.

Thymocyte Development of Humanized Mice Is Promoted by Interactions with Human-Derived Antigen Presenting Cells upon Immunization.

Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205+ dendritic cells (DCs). The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease.

Humanized mice generated by intra-bone marrow injection of CD133-positive hematopoietic stem cells: application to HIV-1 research

Animal models are essential for basic and clinical research on virus diseases. Humanized mice (mice reconstituted with human hematopoietic cells) have been effectively used for various virus studies as small animal models. Studies on human-tropic HIV-1 have also been performed using different humanized mouse models. Various humanized mice have been generated using distinct mouse strains and engraftment methods. These different techniques affect the reconstitution of human hematopoietic cells in individual mice, and in turn the HIV-1 replication in vivo. In this report, we describe the details of the generation method of humanized mice, i.e., severely immunodeficient mice (NSG mice) transplanted with human CD133-positive cells via intra-bone marrow injection (IBMI). It has been shown that the CD133-positive cells are highly capable to generate CD34-positive cells in vivo and IBMI is an excellent methodology for lymphoid and myeloid cell repopulation. In humanized mice transplanted with CD133-positive cells into the bone marrow, human lymphocytes were increased 3 months after the transplantation and a steady increase in CD4-positive cells was observed until 6–8 months after the transplantation. In order to test the utility of our system, CXCR4-tropic and CCR5-tropic HIV-1 clones were intraperitoneally inoculated into the resultant humanized mice 6–8 months after the transplantation. Upon inoculation at the same dose of viruses, the plasma viral load in CCR5-tropic HIV-1-inoculated mice peaked earlier than that in CXCR4-tropic HIV-1-inoculated mice (2–3 weeks vs 5–10 weeks post-inoculation). While a rapid decrease in CD4-positive cells was observed at the peak or prior to the peak of viremia for CXCR4-tropic HIV-1-inoculated mice, CD4-positive cells were gradually decreased in CCR5-tropic HIV-1-inoculated mice. Upon inoculation at the same dose of viruses, a Nef-deleted R5-tropic HIV-1 exhibited retarded growth kinetics in the inoculated mice compared to the parental virus (around 8 weeks vs 2–3 weeks post-inoculation), which appears to reflect the decrease in replication potential in primary cells. Taken all together, in addition to the humanized mice reported so far, our humanized mice generated by transplanting CD133-positive cells with the IBMI method would be an appropriate prototype model for understanding HIV-1 biology in vivo.

Intra-bone marrow injection of magnesium isoglyrrhizinate inhibits inflammation and delays osteoarthritis progression through the NF-κB pathway

ObjectiveOsteoarthritis (OA) presents cartilage damage in addition to chronic inflammation. However, self-recovery of damaged cartilage in an inflammatory environment is not possible. Mesenchymal stem cells (MSCs) in the bone marrow are a source of regenerative repair of damaged cartilage. To date, whether intra-luminal administration of the bone marrow can delay the progression of OA is still unknown. This study, therefore, aimed to explore the role of intra-bone marrow injection of Magnesium isoglycyrrhizinate (MgIG) in delaying the OA progression and to investigate the underlying mechanism.MethodsRabbit OA models were established using the anterior cruciate ligament transection method while a catheter was implanted into the bone marrow cavity. 1 week after surgery, MgIG treatment was started once a week for 4 weeks. The cartilage degradation was analyzed using hematoxylin–eosin staining, Masson’s trichrome staining and Alcian blue staining. Additionally, the pro-inflammatory factors and cartilage regeneration genes involved in the cartilage degeneration and the underlying mechanisms in OA were detected using enzyme-linked immunosorbent assay, quantitative real-time PCR (qRT-PCR) and Western blotting.ResultsThe results of histological staining revealed that intra-bone marrow injection of MgIG reduced degeneration and erosion of articular cartilage, substantially reducing the Osteoarthritis Research Society International scores. Furthermore, the productions of inflammatory cytokines in the bone marrow cavity and articular cavity such as interleukin-1β(IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were inhibited upon the treatment of MgIG. At the same time, the expression of alkaline phosphate, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-telopeptides of type II collagen (CTX-II) in the blood also decreased and was positively correlated. On the contrary, cartilage-related genes in the bone marrow cavity such as type II collagen (Col II), Aggrecan (AGN), and SRY-box 9 (SOX9) were up-regulated, while matrix metalloproteinase-3 (MMP-3) was down-regulated. Mechanistically, MgIG was found to exert an anti-inflammatory effect and impart protection to the cartilage by inhibiting the NF-κB pathway.ConclusionIntra-bone marrow injection of MgIG might inhibit the activation of the NF-κB pathway in the progression of OA to exert an anti-inflammatory effect in the bone marrow cavity and articular cavity, thereby promoting cartilage regeneration of MSCs in the bone marrow, making it a potential new therapeutic intervention for the treatment of OA.

Systematic Safety Evaluation of Cold Plasma-Activated Liquid in Rabbits

Plasma-activated liquid (PAL) can effectively and selectively kill various types of cancer cells both in superficial and deeper tumors. As a promising novel approach to oncotherapy, the safety of PAL is essential in the clinic but has not been thoroughly assessed. In myeloma and blood tumors, the pathogenesis is in the bone marrow cavity. We have therefore evaluated the safety of PAL in New Zealand rabbits by intra-bone marrow injection, and provide a basis of further clinical research and application of PALs. In this study, both a plasma jet and plasma surface were used to treat saline solution, phosphate-buffered solution, and cell culture medium, to produce PAL. Then, oscillograms and optical emission spectra were evaluated to characterize the plasma discharge. Acute toxicity tests and safety evaluation studies were conducted by intra-bone marrow injection of PAL into New Zealand rabbits, while control rabbits received saline only. Body weight, vital organ coefficient, organ appearance, organ histopathology, blood cell and hemoglobin parameters, and blood biochemical indicators were tested on the 30th day after injection. We found that there was no mortality or loss of mobility throughout the experimental period. Acute toxicity tests showed that there were no PAL-related side effects in rabbits receiving the maximum dose of 700 μL PAL. PAL treatments did not affect body weight, organ coefficient, organ appearance, organ histopathology, or blood biochemical indicators. However, the percentage of lymphocytes decreased while the percentage of neutrophil granulocytes increased compared with the control group. In summary, our results indicate that PAL can be safely injected into bone marrow of New Zealand rabbits without significant toxicity.

The Hematopoietic Reconstitution through Intra-Bone-Marrow Injection Using Murine Fetal Liver Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) were considered an effective treatment option for many blood diseases and autoimmune diseases. HSCs can currently be isolated from bone marrow and umbilical cord blood. Due to the shortage of these resources, an alternative source of HSCs preparation were sorted out in this study and the ability of hematopoietic reconstruction were examined using such HSCs obtained from fetal liver.Fetal liver hematopoietic stem cells (FL-HSCs) were isolated from embryos of transgenic mice with an erythoid-specific GFP expression that was driven by a human β-globin gene promoter (HS23-GFP mice). Thus, GFP expression could be used as tracer of marker genes of red blood cells derived from the donor FL-HSCs visually. Single-cell suspension of E13.5 HS23-GFP murine liver was used for Ficoll density gradient separation, and the FL-HSC enriched portion was collected for intra-bone-marrow injection (IBMI) into 6-8 week-old recipient mice post 137Cs γ irradiation treatment. Three doses were tested for injection, i.e. 1×105, 1×106 and 1×107 cells, respectively.Hematopoietic reconstruction ability was observed every week after injection using blood smear and routine blood panel such as RBC, HGB and HCT. Bone marrow smear and pathological sections of liver and spleen of recipient mice were analyzed 8 weeks post FL-HSC transplantation. GFP expressed erythrocyte was captured in the peripheral blood starting one week after HL-HSC transplantation. The ability of hematopoietic reconstruction continued to rise until week 7, and became stable thereafter. Among the three treatment groups, the highest cell number group (1×107 ) had the best hematopoietic restoration, according to the RBC, HGB and HCT data. No abnormality such as lymphocytic infiltration in pathological sections of liver and spleen of recipients were noted. Bone marrow smear of recipient mice showed no difference when comparing to that of the wide type mice. This demonstrated HSC obtained from fetal liver, are capable of reconstructing hematopoietic function after transplantation using IBMI. This could provide an alternative source of HSC for future study of hematopoietic function and for future therapeutic treatment for blood diseases. DisclosuresNo relevant conflicts of interest to declare.

Magic-TT (Magnetism-induced cell target transplantation) Improves the Effect of Intra-Bone Marrow Transplantation Resulting in Rapid Hematopoietic Reconstitution in Allogenic Murine Transplantation Model

Background: Intra-bone marrow transplantation has been proven to be efficient in improving the homing of transplanted cells, resulting in faster hematopoietic reconstitution and a reduction in severity of GVHD. However, because of staggered blood vessels in bone, many transplanted cells leak out to peripheral circulation. In our previous study (Blood 2015 126:5404), it has been initially proven that MagIC-TT could enhance target transplantation to femurs by flow cytometry and pathological examination. After that, by bioluminescent imagine system, we further proved that most intra-bone marrow transplanted cells moved to lung immediately without MagIC-TT (Figure 1 A); while the cells were retained in the bone marrow with MagIC-TT (Figure 1 B), but if the magnetic field withdrawal 1 hour after, the cells also moved to the lung quickly(Figure 1 C). In this study, the therapeutic effect of intra-bone marrow transplantation combined with MagIC-TT was explored in allogenic murine transplantation model.MethodsMice and cell preparation: Sixty female BALB/c mice with specific pathogen free (SPF) were randomly separated in 4 groups (16 and 14 mice in 5×10e6M and 1×10e7 M group respectively; 16 and 14 mice in 5×10e6W and 1×10e7 W group respectively, M/W referring to MagIC-TT/non-MagIC-TT). The recipients received 7.5Gy irradiation before transplantation. Twelve SPF C57BL/6 CAG-eGFP transgenic mice were used as donors. The bone marrow were magnetized by nanoparticles and then adjusted to the concentration of 5×10e6/20μL and 1×10e7/20μl for use. Every mouse was injected with 20μL, 5×10e6 or 1×10e7 cells.Intra-bone marrow injection and fixed magnetic field After anesthetizing the mice and disinfecting the knee joint, the femoral puncture was operated with the medulla-puncture needle invented by our team (Chinese patent No.201620090904.2). In M groups, special magnetic fields were applied to the femurs of mice during and after injection. The effects of intra-bone marrow injection had been proven by X-ray and bioluminescent imaging system, etc .The therapeutic effects The therapeutic effects were determined by the survival of mice, the percentage of GFP+ cells in peripheral blood, blood routine test etc. to evaluate the hematopoietic reconstitution.ResultsSurvival: The survival rate was 66.7% and 33.3% in M groups and W groups on the 40th day after transplantation (P=0.096) (Figure 2 A), which was 62.5% and 71.4% in 5×10e6M and 1×10e7M, 37.5% and 28.6% in 5×10e6W and 1×10e7W, respectively(Figure 2 B).Donor cells percentage in peripheral blood (Figure 3 A): Flow cytometry analysis was performed on the 5th, 13rd, 17th, and 25thday after transplantation. The percentage of donor GFP+ cells grew much faster on d5~d13 in M groups than that in W groups (P<0.05). Pairwise comparisons showed that 10e7M group grew faster than 10e7W (P<0.05), donor GFP+ cells in 5×10e6M grew even faster than those in both 5×10e6W and 1×10e7W (P<0.05). No significant difference was found between 5×10e6M and 1×10e7M.Blood Routine Test: Generally, WBC in M groups reached growth peak on d9-13 vs d13-17 in W groups, and the WBC number of M groups are higher than those in W group (P<0.05) (Figure 3B). This trend also existed for HGB and PLT, but not as significant as WBC (Figure 3 C, 3 D) .ConclusionMagIC-TT can improve the effect of intra-bone marrow transplantation by avoiding transplanted cells leakage to peripheral circulation, resulting in faster hematopoietic reconstitution in allo-murine transplantation model. The mechanism of MagIC-TT and its relation with reduction of complication are under further study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reprogrammed mesenchymal stem cells derived from iPSCs promote bone repair in steroid-associated osteonecrosis of the femoral head

BackgroundCellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations.MethodsIn the present study, the bone marrow samples of patients with ONFH (n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation.ResultsThe morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head.ConclusionReprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.

Cotransplantation of Umbilical Cord Mesenchymal Stem Cells Promotes the Engraftment of Umbilical Cord Blood Stem Cells in Iron Overload NOD/SCID Mice

Iron overload aggravates the difficulty of umbilical cord blood (UCB) stem cell engraftment and reduces the survival of patients undergoing hematopoietic stem cell (HSC) transplantation. Mesenchymal stem cells (MSCs) have been suggested to have a significant role in HSC engraftment. This study aimed to determine the effect of intra-bone marrow (IBM) and i.v. cotransplantation of UBC mononuclear cells (MNCs) and umbilical cord (UC) MSCs on engraftment and hematopoietic recovery in an iron overload hematopoietic microenvironment. The iron overload model was established by dose-escalation intraperitoneal injection of iron dextran in NOD/SCID mice. Iron deposition in the bone marrow, heart, and liver was examined using hematoxylin and eosin (H&E) staining. Serum levels of ferritin and iron status in the liver were measured. The iron overload NOD/SCID mice were sublethally irradiated and divided into 5 groups for transplantation: (1) control group; (2) IBM+ group: IBM injection of combined UCB-MNCs/UC-MSCs; (3) IV+ group: i.v. injection of combined UCB-MNCs/UC-MSCs; (4) IBM group: IBM injection of only UCB-MNCs; and (5) IV group: i.v. injection of UCB-MNCs. At 6 weeks after transplantation, the human CD45+ cells in the bone marrow were analyzed by flow cytometry. The semiquantitative analysis of vascular endothelial growth factor (VEGF-A), osteopontin (OPN), and stromal cell-derived factor-1a (SDF-1a) were examined by immunohistochemical staining (IHC). Compared with the IBM and IV groups, the survival rate and the percentages of human CD45+ cells and CD34+ cells and colony-forming units (CFU) in bone marrow were elevated in the IBM+ and IV+ groups. In addition, the levels of VEGF-A, OPN, and SDF-1a in bone marrow were all higher in the IBM+ and IV+ groups. Our data show that IBM and i.v. cotransplantation of UC-MSCs can improve the engraftment and proliferation of UCB-MNCs in iron overload NOD/SCID mice. The increased expression of VEGF-A, OPN, and SDF-1a in the bone marrow may be involved in improving the hematopoietic microenvironment and promoting the implantation of human UCB stem cells in the bone marrow with iron overload.

Lentivirus-delivered ACE siRNA rescues the impaired peak bone mass accumulation caused by prenatal dexamethasone exposure in male offspring rats.

Angiotensin I converting enzyme (ACE) is a major component of the renin-angiotensin system (RAS). Our previous study demonstrated that activated bone RAS was associated with low peak bone mass induced by prenatal dexamethasone exposure (PDE) in male offspring rats. However, we did not determine whether the inhibition of ACE expression could rescue PDE-induced low peak bone mass. In the present study, we treated pregnant Wistar rats with dexamethasone (0.2mg/kg.d) on gestational days 9-20 and obtained eight weeks old male offspring rats. Some of the offspring rats from the PDE group were injected lentivirus delivered-ACE siRNA (LV-ACE siRNA) through the intra-bone marrow for 4weeks. We found that the intra-bone marrow injection of LV-ACE siRNA rescued the impaired peak bone mass accumulation caused by PDE in male offspring rats. Moreover, LV-ACE siRNA ameliorated PDE-induced inhibition of osteogenesis and alleviated PDE-induced RAS activation in the bone tissues in vivo. Our in vitro findings further confirmed that LV-ACE siRNA reversed the suppressed osteogenic differentiation caused by dexamethasone, which can be attributed to alleviated RAS activation. In conclusion, LV-ACE siRNA rescued impaired peak bone mass accumulation caused by PDE through alleviation of local bone RAS activation in male offspring rats.

Short-Term exposure of umbilical cord blood CD34+ cells to human platelet lysate and cytokines enhances engraftment.

Intra bone marrow (IBM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic progenitor stem cell (HSPC) transplantation and thus increases the number of HSPC that engraft. Despite physical delivery into the bone marrow cavity, many donor cells are rapidly redistributed by vascular perfusion. Thus, the objective of our study was to evaluate the ability of human platelet lysates (hPL) to improve HSPC retention into the bone marrow and consequently to improve engraftment. HSPC were isolated from human umbilical cord blood. HSPC were seeded in the wells of a 24-well microplate and exposed to increasing concentrations of hPL with or without cytokines for 24 hours. Following priming, HSPC cells chemotaxis to rhSDF-1 was determined in vitro and engraftment in NSG mice was evaluated. Priming of cord blood CD34+ cells to a combination of hPL and cytokines resulted in a significant increase (up to 3-fold) in the expression of the CD34 antigen on HSPC. This effect was closely correlated to a significantly increased (up to 7-fold) migration toward a rhSDF-1 concentration gradient. In addition, IBM injection of CD34+ cells previously primed with hPL+cytokines into NSG mice showed significantly increased engraftment as measured by human platelet numbers, human CD45 and human CD34+ cells for unprimed and primed cells, respectively. The use of hPL + cytokines as a short-term priming treatment for UCB could be an advantageous strategy to improve clinical outcomes following IBM injection.

Loss of p53 in mesenchymal stem cells promotes alteration of bone remodeling through negative regulation of osteoprotegerin

p53 plays a pivotal role in controlling the differentiation of mesenchymal stem cells (MSCs) by regulating genes involved in cell cycle and early steps of differentiation process. In the context of osteogenic differentiation of MSCs and bone homeostasis, the osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) axis is a critical signaling pathway. The absence or loss of function of p53 has been implicated in aberrant osteogenic differentiation of MSCs that results in higher bone formation versus erosion, leading to an unbalanced bone remodeling. Here, we show by microCT that mice with p53 deletion systemically or specifically in mesenchymal cells possess significantly higher bone density than their respective littermate controls. There is a negative correlation between p53 and OPG both in vivo by analysis of serum from p53+/+, p53+/−, and p53−/− mice and in vitro by p53 knockdown and ChIP assay in MSCs. Notably, high expression of Opg or its combination with low level of p53 are prominent features in clinical cancer lesion of osteosarcoma and prostate cancer respectively, which correlate with poor survival. Intra-bone marrow injection of prostate cancer cells, together with androgen can suppress p53 expression and enhance local Opg expression, leading to an enhancement of bone density. Our results support the notion that MSCs, as osteoblast progenitor cells and one major component of bone microenvironment, represent a cellular source of OPG, whose amount is regulated by the p53 status. It also highlights a key role for the p53-OPG axis in regulating the cancer associated bone remodeling.

Phase I Study of Cord Blood Transplantation with Intra-Bone Marrow Injection of Mesenchymal Stem Cells

Phase I Study of Cord Blood Transplantation with Intra-Bone Marrow Injection of Mesenchymal Stem Cells

Co-Transplantation of Ucbmn with UC-MSC Enhances Engraftment of Ucbmn in Iron Overload NOD/SCID Mice

Background: Umbilical cord blood (UCB)-derived hematopoietic stem cells (HSC) are ideal for allogeneic HSCT when a compatible donor is unavailable. However, the insufficient number of HSCs per UCB unit delays engraftment and may lead to graft failure and/or mortality. Besides, transfusion-induced iron overload has been reported to be involved in the delayed engraftment of HSC. Previously mesenchymal stem cells (MSC) have been implicated in playing a significant role in HSC engraftment. This study aimed to determine the effect of co-transplantation of umbilical cord mesenchymal stem cells (UC-MSC) on engraftment and hematopoietic recovery.Methods: The iron overload NOD/SCID mice were induced by dose-escalation injection of iron dextran. And the sublethally irradiated iron overload NOD/SCID mice were divided into four groups: (1) intravenous injection of normal saline (control group, n=8), (2) intravenous injection of umbilical cord mesenchymal MN) alone (n=10), (3) intravenous injection of UCBMN and UC-MSC (n=10), and (4) intrafemoral injection of UCBMN and UC-MSC (n=12). Six weeks after injection of UC, the bone marrow (BM) was harvested and analyzed. The semi-quantitative analysis of cytokines including SDF-1a, OPN, and VEGF-α were examined by immunohistochemistry.Results: As a result, iron deposits in bone liver, BM and spleen. In the BM of control group, no human CD45+ cells were found. Via the intra-bone marrow (iBM) injection, the percentage of human CD45+ cells was 9.86% ±1.86% (n=10) in the presence and 3.18% ±0.98% (n=6) in the absence of MSC, respectively, via the tail vein, the percentage of human CD45+ cells was 3.5%±0.94%. In additional, the levels of SDF-1a, OPN, and VEGF-a were all significantly higher in The mice that received MSC showed significantly enhanced engraftment in all three experiments (P=0.000).Conclusion: Co-transplantation of UC-MSC is effective for improving engraftment in iron overload NOD/SCID mice. The point underlying the positive outcome may relate to cytokines including SDF-1a, OPN, and VEGF-a secreted by UC-MSC, which has been indicated to play a crucial part in homing and engraftment. DisclosuresNo relevant conflicts of interest to declare.

Updated human hematopoietic stem cell findings: purification of human hematopoietic stem cells and elucidation of their hierarchy

Hematopoietic stem cell (HSC) biology is a current topic of interest having significant implications for clinical HSC transplantation and basic HSC research. It was long believed that the most primitive HSCs in mammals, including those in mice and humans, were CD34 antigen-positive (CD34+) cells. However, Nakauchi et al. reported that murine long-term lymphohematopoietic reconstituting HSCs were lineage marker-negative (Lin-) c-kit+Sca-1+CD34-low/negative (CD34low/-), known as CD34low/-KSL cells. We have previously identified very primitive human cord blood (CB) -derived CD34-negative (CD34-) severe combined immunodeficiency (SCID) -repopulating cells (SRCs) utilizing an intra-bone marrow injection method and have proposed a new concept that CD34-SRCs (HSCs) reside at the apex of human HSC hierarchy. Recently, we developed an ultra-high-resolution purification method using the two positive/enrichment markers CD133 and GPI-80 and succeeded in purifying CD34+/-HSCs on a single-cell level. On the basis of these data, we propose a revised roadmap for the commitment of human CD34-HSCs. This review updates the concept of the stem cell nature of human CB-derived primitive CD34+/-HSCs.

CD34-negative hematopoietic stem cells show distinct expression profiles of homing molecules that limit engraftment in mice and sheep.

We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34-) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin- or 18Lin-) CD34+/- cells from human CB in mice and sheep. Both 13Lin- and 18Lin- CD34+ cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin- nor 18Lin- CD34- cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin- and 18Lin- CD34- cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34- HSCs is significantly more limited than that of CD34+ HSCs. As for 18Lin-, CD34- HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34- HSCs could thus explain in part their reduced ability to home to the BM niche.

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34– severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin–CD34+/–MPL+/– cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/–MPL+/– cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/– and CD34–MPL– cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/–MPL– cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/– SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34– SRCs generate CD34+CD38–CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34–MPL– SRCs reside at the apex of the human HSC hierarchy.

Isolation of Single Human Cord Blood-Derived CD34-Negative Hematopoietic Stem Cells (HSCs) Residing at the Apex of the Human HSC Hierarchy

Background. We identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). Recently, we identified CD133 as a positive marker of human CB-derived CD34+/- SRCs. Limiting dilution analyses (LDAs) demonstrated that the frequencies of SRCs in the 18Lin-CD34+/-CD133+ fractions were 1/100 and 1/140, respectively (Leukemia 2014:28;1308). We then identified glycosylphosphatidylinositol-anchored surface protein (GPI-80) as a useful enrichment marker of human CB-derived CD34+/- SRCs and succeeded in highly purifying primitive human CD34+/- SRCs to the level of 1/20 cells.Aim. We attempted to purify CD34+/- SRCs to the single-cell level using CD133 and GPI-80 in order to precisely characterize the CD34- HSCs in comparison to CD34+ HSCs.Materials and Methods. We first developed an ultra-high-resolution purification method using two positive/enrichment markers for CD34+/- SRCs, including CD133 and GPI-80. We sorted 18Lin-CD34+CD38-CD133+GPI-80+ (34+38-133+80+)and 18Lin-CD34- CD133+GPI-80+ (34-133+80+)cells by FACS. Thereafter, these two fractions of cells were transplanted by the IBMI technique into NOG/NSG mice to investigate their long-term repopulating capacities. Finally, we performed single-cell transplantations. We then analyzed the gene expression profiles of single 34+38-133+80+ and 34-133+80+ cells using a BioMark System (Fluidigm). In parallel, we performed microarray experiments to analyze their gene expression profiles.Results. Approximately 14% of 34+38-133+ cells and 8% of 34-133+ cells expressed GPI-80. These highly purified cells showed very immature blast-like morphologies. These two fractions of cells were then transplanted into NOG/NSG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. All of the mice that received 200 34+38-133+80+ (n=25)and 34-133+80+ (n=23) cells were repopulated with human CD45+ cells, including CD34+, CD19+ and CD33+ cells. Almost all of the secondary transplanted mice showed comparable human CD45+ cell reconstitution with multi-lineage differentiation. An LDA demonstrated that the frequencies of CD34+/- SRCs in the 34+38-133+80+ and 34-133+80+ cells were 1/5 and 1/8, respectively. Interestingly, 13.2% (7/53) of the recipient NSG mice that received single 34+38-133+80+ cells displayed distinct human CD45+ cell reconstitution (0.5%-44.2%) with multi-lineage human cell repopulation at 20 weeks after transplantation. In contrast, 6.6% (5/76) of the recipient NSG mice that received single 34-133+80+ cells displayed distinct human CD45+ cell reconstitution (0.6%-30.1%) with multi-lineage human cell repopulation at 20 weeks after transplantation. Both groups of reconstituted mice showed secondary reconstitution with multi-lineage differentiation. These results indicated that individual 34+38-133+80+ and 34-133+80+ cells extensively self-renew. We then analyzed the gene expression profiles of these two types of SRCs at the single-cell level. A principle component analysis showed that the gene expression profiles of individual CD34+ and CD34- SRCs were clearly different. Both SRCs expressed high levels of HSC maintenance genes, including RUNX1 and BMI1. Very interestingly, CD34+ SRCs expressed a high level of IFITM1, IKZF1 and ETV6. In contrast, CD34- SRCs expressed higher levels of EZH2 than CD34+ SRCs. Furthermore, a gene set enrichment analysis demonstrated that 34+38-133+80+ cells expressed higher levels of genes related to cell adhesion, chemokine signaling, and trans-endothelial migration, whereas 34-133+80+ cells expressed higher levels of genes related to developmental maturation. Very interestingly, 34-133+80+ cells expressed lower levels of interferon signal-related genes, including STAT1, IFTM1, IFTM3, Ddx58, IFI44, CCL5 and CXCL10, than 34+38-133+80+ cells. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two types of CD34+/- SRCs.Conclusion. Wedeveloped an ultra-high-resolution purification method using two markers for CD34+/- SRCs, including CD133 and GPI-80. This precise single cell-based analysis allows us to map CD34- SRCs (HSCs) at the apex of the human HSC hierarchy. DisclosuresNo relevant conflicts of interest to declare.

Intrabone marrow injection enhances placental mesenchymal stem cellmediated support of hematopoiesis in mice.

In order to determine the synergistic effects of human placental mesenchymal stem cells (PMSCs) on hematopoiesis in vivo, we compared the intrabone marrow injection (IBMI) with the conventional intravenous injection (IVI). C57BL/6 recipient mice conditioned with lethal doses of irradiation were transplanted with bone marrow mononuclear cells (MNCs) and bone marrow-derived mesenchymal stem cells (BMSCs) from BALB/c mice by IBMI or IVI. NOD/SCID recipient mice conditioned with sublethal doses of irradiation were transplanted with human umbilical cord blood MNCs (UCB-MNCs) and PMSCs by IBMI or IVI. The number of hematopoietic cells was significantly higher in mice transplanted with BMSCs by IBMI than in those transplanted by IVI in a murine transplantation model (BALB/c→C57BL/6). Moreover, the percentage of human hematopoietic cells in the tibiae of the NOD/SCID mice that were transplanted with PMSCs plus UCB-MNCs was higher than that in mice transplanted with UCB-MNCs alone. In addition, in mice that were transplanted with PMSCs, PMSCs injected by IBMI were more efficient than those injected by IVI. Our results not only elucidated the role of PMSCs in promoting hematopoiesis, but also revealed the therapeutic potential of the combination of PMSCs and IBMI in transplantation.