Migration Of Bone Marrow Stem Cells Research Articles

Alkaline activation of endogenous latent TGFβ1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFβ1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFβ1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFβ1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Exercise Inhibits Doxorubicin-Induced Damage to Cardiac Vessels and Activation of Hippo/YAP-Mediated Apoptosis

Simple SummaryWe developed a juvenile mouse cardiotoxicity model and demonstrated that doxorubicin treatment led to cardiac vascular damage involving a decrease in vascular endothelial cells and pericytes. By contrast, cardiac vessels in mice treated with exercise (Ex) during Dox treatment had no change in vascular endothelial cells or pericyte coverage. Here we show that exercise during Dox induced the migration of bone marrow (BM) stem cells into the heart with their differentiation into vascular endothelial cells and pericytes. These data define a new mechanism of Dox-induced cardiotoxicity and suggest that Ex inhibits vascular damage by promoting bone marrow (BM) stem cell-mediated cardiac vessel repair. We also show that Ex inhibits Hippo-YAP signaling-mediated apoptosis in cardiomyocytes. Ex may be an effective intervention strategy. Targeting Hippo-YAP signaling may also offer another novel therapeutic option to protect the heart.Dose-related cardiomyopathy is a major side effect following doxorubicin (Dox). To investigate whether exercise (Ex)-induced vasculogenesis plays a role in reducing Dox-induced cardiotoxicity, GFP+ bone marrow (BM) cells from GFP transgenic mice were transplanted into wild-type mice. Transplanted mice were treated with Dox, Ex, Dox+Ex, or control. We found Dox therapy resulted in decreased systolic and diastolic blood flow, decreased ejection fraction and fractional shortening, and decreased vascular endothelial cells and pericytes. These abnormalities were not seen in Dox+Ex hearts. Heart tissues from control-, Ex-, or Dox-treated mice showed a small number of GFP+ cells. By contrast, the Dox+Ex-treated hearts had a significant increase in GFP+ cells. Further analyses demonstrated these GFP+ BM cells had differentiated into vascular endothelial cells (GFP+CD31+) and pericytes (GFP+NG2+). Decreased cardiomyocytes were also seen in Dox-treated but not Dox+Ex-treated hearts. Ex induced an increase in GFP+c-Kit+ cells. However, these c-Kit+ BM stem cells had not differentiated into cardiomyocytes. Dox therapy induced phosphorylation of MST1/2, LATS1, and YAP; a decrease in total YAP; and cleavage of caspase-3 and PARP in the heart tissues. Dox+Ex prevented these effects. Our data demonstrated Dox-induced cardiotoxicity is mediated by vascular damage resulting in decreased cardiac blood flow and through activation of Hippo-YAP signaling resulting in cardiomyocyte apoptosis. Furthermore, Ex inhibited these effects by promoting migration of BM stem cells into the heart to repair the cardiac vessels damaged by Dox and through inhibiting Dox-induced Hippo-YAP signaling-mediated apoptosis. These data support the concept of using exercise as an intervention to decrease Dox-induced cardiotoxicity.

Abstract 5422: Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity

Abstract Doxorubicin (Dox) is one of the most effective drugs for the treatment of childhood cancer patients. However, there is a correlation between Dox dose/intensity and cardiotoxicity. We developed a juvenile mouse cardiotoxicity model and showed that Dox treatment affected the cardiac vessel morphology characterized by punctate vessels with decreased pericytes and endothelial cells. Cardiac blood flow and function (as quantified by echocardiography [Echo]) were also compromised. These abnormalities were not seen in mice treated with exercise (Ex) during Dox. We also demonstrated increased c-kit+ cells in the hearts of mice treated with Dox + Ex. To determine the origin of these c-kit+ cells we performed a bone marrow (BM) transplant using GFP+ BM cells from C57BL/6Tg(UBC-GFP)30Scha/J mice into C57BL/6 WT mice treated with Busulfan and anti-CD4 and anti-DC8 Abs. BM engraftment was documented 4 wks post-transplant. Transplanted mice were treated with Dox twice/wk x 2 wks; Dox + Ex, or control (no treatment). Cardiac function was assessed after treatment by Echo and the heart tissues were evaluated by immunohistochemistry (IHC) for cells expressing GFP (denoting cells derived from the BM); GFP+/NG2+ or GFP+/CD31+ (BM cells differentiated into pericytes or endothelial cells respectively). Echo data confirmed decreased cardiac function in Dox but not Dox + Ex-treated mice. Few GFP+ cells were found in the hearts from control or Dox-treated mice. By contrast, mice treated with Dox + Ex showed a significant increase in GFP+ cells indicating that exercise had induced the migration of BM cells into the heart. Co-localization of GFP/NG2 and GFP/CD31 indicated that these BM stem cells had differentiated into both endothelial cells and pericytes, and that these cells were incorporated into the cardiac vessels. Few GFP+cTnI+ cells were observed in the heart tissues from mice treated with Ex + Dox, indicating that the BM stem cells had not differentiated into cardiomyoctes. These studies suggest that acute Dox-induced cardiac damage may be partially mediated by decreased blood flow secondary to damaged vessels with decreased vascular endothelial cells and pericytes. Ex may inhibit this effect by inducing the migration of BM stem cells to repair the damaged vessels. We also showed that Dox therapy induced phosphorylation of MST1/2, LATS1 and YAP, decreased total YAP, and induced cleavage of caspase-3 and PARP. These changes were not seen in Dox + Ex hearts suggesting that Ex protected cardiomyocytes from Dox-induced apoptosis by suppressing Hippo-YAP signaling. Taken together, our data demonstrated that the protective effect of Ex may be mediated by restoring cardiac blood flow which may in turn inhibit Dox-induced Hippo-YAP signaling-mediated cardiomyocyte apoptosis. These data support the concept that exercise interventions have the potential to decrease Dox-induced cardiac damage and morbidity in childhood cancer patients. Citation Format: Rong-Hua Tao, Masato Kobayashi, Fei Wang, Yuanzheng Yang, Eugenie S. Kleinerman. Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5422.

MicroRNA-150 Modulates Adipogenic Differentiation of Adipose-Derived Stem Cells by Targeting Notch3.

MicroRNAs (miRNAs) influence stem cell functions, including mobilization, proliferation, and differentiation. miR-150 is abundantly expressed in monocytes. Knockdown of miR-150 promotes bone marrow stem cell migration. The role of miR-150 in adipose-derived stem cells (ADSCs) is unclear. In this study, the effects of miR-150 on adipogenic differentiation and proliferation of ADSCs were investigated. ADSCs were isolated from the inguinal adipose tissue of wild-type (WT) and miR-150 knockout (KO) mice and were induced for adipogenic differentiation. The miR-150 level was detected by real-time PCR. ADSCs were transfected by miR-150 or small-interfering RNA (siRNA) of Notch3. MTT assay and colony formation assay were performed in miR-150 knockdown and control ADSCs. Real-time PCR showed that miR-150 was expressed in ADSCs. miR-150 knockdown significantly decreased the capacity of adipogenic differentiation of ADSCs, as compared with their counterparts from WT mice. It is intriguing that the overexpression of miR-150 significantly increased C/EBPα and PPAR-γ expression and lipid formation in ADSCs with adipogenic induction. Overexpression of miR-150 significantly decreased Notch3 expression in ADSCs compared with the control groups. Furthermore, Notch3 inhibition promoted the adipogenic differentiation in ADSCs. miR-150 also suppressed proliferation potential and the expression of Nanog in ADSCs. In summary, this study demonstrates, for the first time, that miR-150 promotes adipogenic differentiation and inhibits proliferation of ADSCs. miR-150 regulates adipogenic differentiation of ADSCs, likely mediated by the downregulation of Notch3.

Strontium‑containing α‑calcium sulfate hemihydrate promotes bone repair via the TGF‑β/Smad signaling pathway.

Calcium phosphate-based bone substitutes have been widely used for bone repair, augmentation and reconstruction in bone implant surgery. While some of these substitutes have shown excellent biological efficacy, there remains a need to improve the performance of the current calcium phosphate-based bone substitutes. Strontium ions (Sr) can promote new osteogenesis, inhibit osteoclast formation and increase osteoconductivity. However, the therapeutic effect and mechanism of strontium-containing α-calcium sulfate hemihydrate (Sr-CaS) remains unclear. The present study created bone injuries in rats and treated the injuries with Sr-CaS. Then Cell Counting Kit-8, soft agar colony formation, flow cytometry, Transwell and Alizarin Red staining assays were performed to assess the bone cells for their proliferation, growth, apoptosis, invasion, and osteogenic differentiation abilities. The bone reconstructive states were measured by the microCT method, hematoxylin and eosin staining and Masson staining. Bone-related factors were analyzed by the reverse transcription-quantitative PCR assay; transforming growth factor (TGF)-β, mothers against decapentaplegic homolog (Smad)2/3 and β-catenin expression was measured by western blot analysis and osteocalcin (OCN) expression was assessed by immunohistochemistry. Sr-CaS did not significantly affect the proliferation and apoptosis of bone marrow stem cells (BMSCs), but did accelerate the migration and osteogenic differentiation of BMSCs in vitro. Sr-CaS promoted bone repair and significantly increased the values for bone mineral density, bone volume fraction, and trabecular thickness, but decreased trabecular spacing in vivo in a concentration-dependent manner. In addition, Sr-CaS dramatically upregulated the expression levels of genes associated with osteogenic differentiation (Runt-related transcription factor 2, Osterix, ALP, OCN and bone sialoprotein) both in vitro and in vivo. Sr-CaS also increased Smad2/3, TGF-β and phosphorylated-β-catenin protein expression in vitro and in vivo. These results indicated that materials that contain 5 or 10% Sr can improve bone defects by regulating the TGF-β/Smad signaling pathway.

Co-culture of bone marrow stem cells and macrophages indicates intermediate mechanism between local inflammation and innate immune system in diabetic periodontitis.

Diabetic periodontitis (DP), which has been shown to cause alveolar bone loss, is among the most common complications associated with diabetes. The precise mechanisms underlying alveolar bone loss in patients with DP remain unclear. Therefore, the present study established a co-culture system of bone marrow stem cells (BMSCs) and macrophages, in order to investigate the potential mechanisms underlying DP-associated alveolar bone loss in vitro. In addition, Porphyromonas gingivalis (PG) periodontal infection and high glucose levels were used to induce DP in mice. The present study evaluated the protein expression levels of various chemokines and the migration of BMSCs and macrophages. The protein expression levels of extracellular signal-regulated kinase 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) were significantly increased in the BMSCs exposed to high glucose and PG, which may have been due to the activation of MAPK. In addition, DP induction in mice was associated with the release of chemokine (C-C motif) ligand 2 (CCL2) from BMSCs and the secretion of chemokine (C-C Motif) receptor 2 (CCR2) and tumor necrosis factor-α from macrophages, which was associated in turn with enhanced adhesion and chemotaxis of macrophages. The results of the present study suggested that DP led to the upregulation of CCL2 in the periodontal tissues and enhanced macrophage infiltration via the CCL2/CCR2 axis, which in turn promoted alveolar bone loss.

Effect of Ermiao Fang with Xixin (Herba Asari Mandshurici) on bone marrow stem cell directional homing to a focal zone in an osteoarthritis rat model

ObjectiveTo investigate the effects of Ermiao Fang (EM) with medical guide Xixin (Herba Asari Mandshurici) (HAM) on bone marrow stem cell migration to a focal zone in osteoarthritis (OA) rats. MethodsOA rats were induced by arthrectomy and assigned to sham-operated, model, EM, or EM plus HAM groups. All rats were injected with recombinant human granulocyte colony-stimulating factor 30 μg • kg−1 • d−1 for 7 days and treated with EM or EM plus HAM at 1.6 or 1.9 g • kg−1 • d−1 for 3 or 6 weeks, respectively. Chondrocyte apoptosis and cartilage matrix components were tested by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay and special staining. Levels of interleukin-1 beta (IL-1β) tumor necrosis factor alpha (TNF-α) nitric oxide (NO), and inducible nitric oxide synthase (iNOS) in serum were detected by enzyme-linked immunosorbent assay or radioimmunoassay. Matrix metalloproteinases (MMPs)-13, tissue inhibitors of metalloproteinases (TIMPs)-1, Bromodeoxyuridine (BrdU), cluster of differentiation 34 (CD34), and stromal cell-derived factor 1 (SDF-1) were measured by immunohistochemical assay. ResultsThe EM and EM plus HAM groups had significantly less cartilage damage and synovium inflammation the model group. Moreover, the EM and EM plus HAM groups had less chondrocyte apoptosis and more proteoglycan and collagen content than the model group. The EM and EM plus HAM groups had obviously higher MMPs-13 and TIMPs-1 expression in the cartilage than the model group. Moreover, the two formula groups had less release of IL-1β, TNF-α, NO, and iNOS than model group. Importantly, the expressions of BrdU, CD34, and SDF-1 in cartilage were significantly higher in the EM and EM plus HAM-Medtreated rats than model group. Notably, the EM plus HAM treatment seemed to have the greatest effects. ConclusionsHAM improves the therapeutic effects of EM on OA rats by enhancing BMSC directional homing to the focal zone.

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Long Course Hyperbaric Oxygen Stimulates Neurogenesis and Attenuates Inflammation after Ischemic Stroke

Several studies have provided evidence with regard to the neuroprotection benefits of hyperbaric oxygen (HBO) therapy in cases of stroke, and HBO also promotes bone marrow stem cells (BMSCs) proliferation and mobilization. This study investigates the influence of HBO therapy on the migration of BMSCs, neurogenesis, gliosis, and inflammation after stroke. Rats that sustained transient middle cerebral artery occlusion (MCAO) were treated with HBO three weeks or two days. The results were examined using a behavior test (modified neurological severity score, mNSS) and immunostaining to evaluate the effects of HBO therapy on migration of BMSCs, neurogenesis, and gliosis, and expression of neurotrophic factors was also evaluated. There was a lower mNSS score in the three-week HBO group when compared with the two-day HBO group. Mobilization of BMSCs to an ischemic area was more improved in long course HBO treatments, suggesting the duration of therapy is crucial for promoting the homing of BMSCs to ischemic brain by HBO therapies. HBO also can stimulate expression of trophic factors and improve neurogenesis and gliosis. These effects may help in neuronal repair after ischemic stroke, and increasing the course of HBO therapy might enhance therapeutic effects on ischemic stroke.

Transplanted xenogenic bone marrow stem cells survive and generate new bone formation in the posterolateral lumbar spine of non-immunosuppressed rabbits

Bone marrow stem cells (BMSCs) are pluripotent cells that have been used to facilitate bone repair because of their capability of differentiating into osteoblasts. However, it is well known that the number of BMSCs with osteogenic potential decreases in patients with old age, osteoporosis, and metabolic diseases. In such conditions, xenogenic BMSCs may provide an alternative to autologous BMSCs. In the current study, we investigated the potential of transplanted xenogenic BMSCs to survive and generate new bone formation in the posterolateral lumbar spine of non-immunosuppressed rabbits. The BMSCs were obtained from bilateral femurs of four male rats, cultured and expanded in medium with osteoinduction supplement. The BMSCs (1,000,000 cells) of male rats loaded onto 5 cc compression resistant matrix (CRM; Medtronic Sofamor Danek, USA) were implanted bilaterally onto the L4-5 intertransverse processes of 16 female rabbits (xenogenic BMSCs + CRM group). The 16 female rabbits that received 5 cc CRM alone were used as controls (CRM alone group). To exclude the possibility of migration of BMSCs from the transverse processes of the recipient rabbits, we did not decorticate the transverse processes. No rabbits received any immunosuppressive medications during the experiment. Four rabbits each in both of the experimental and control groups were killed at 1, 2, 4, and 6 months postimplantation, and the lumbar spine underwent radiological and histological analyses for evaluation of new bone formation. The polymerase chain reaction (PCR) for Sry gene (Y-chromosome-specific marker) was used to evaluate the survival of transplanted xenogenic BMSCs. The expression of Sry gene was clearly identified in the lumbar spines of all the 16 rabbits in the xenogenic BMSCs + CRM group at 1-6 months postimplantation. Serial plain radiographs showed gradual resorption of CRM; however, it was difficult to clearly identify the presence of new bone formation due to the radiopacity of the remaining CRM. Histologically, mature lamellar and woven bone with osteoblasts and osteocytes were identified in all eight rabbits in the xenogenic BMSCs + CRM group at 4 and 6 months postimplantation, but in none of the eight rabbits at 1 and 2 months postimplantation. None of CRM alone group showed new bone formation at 1-6 months postimplantation. Mild-to-moderate infiltration of inflammatory cells was identified around the CRM carriers in both the groups. No post-operative wound infection was found in either group. Our results indicate that xenogenic BMSCs loaded onto CRM survive and generate new bone formation when placed into the posterolateral lumbar spine of rabbits without immunosuppression. To determine if a solid fusion can be achieved with such techniques, further studies are needed to investigate the appropriate dose of xenogenic BMSCs, amounts of CRM, and the requisite incubation time.

Depletion of glutathione attenuates response of bone marrow stem cells to stromal cell‐derived factor ‐1α

In adult organisms, stem cells, found primarily in the bone marrow and in low numbers in other organs, are mobilized to the injured tissue where they participate in the repair process. Stromal cell-derived factor -1a (SDF-1a) is critical for stem cell trafficking during injury. Very little is known about how nutrition affects this process. Our objectives were to determine how Glutathione (GSH) depletion, observed with aging and in numerous chronic disease states, affects the functional response of bone marrow cells to SDF-1a. Bone marrow stem cells (BMSC) from 8 week old C57BL/6J mice were isolated, and were cultured for 24 hours in buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. SDF-1a (120 ng/ml) dependent chemotaxis was determined in viable cells, over 3 hours, using a modified Boyden chamber method. We found a 5 fold decline in migration, in cells depleted of GSH compared to control. Our data show that GSH is important in the directional migration of BMSC to SDF-1a and indicate that signal transduction pathways initiated by SDF-1a may be redox sensitive. Therefore, in states of chronic oxidative imbalance, stem cell response to SDF-1a may be attenuated slowing tissue regeneration and repair. As nutrition can impact levels of GSH, dietary control of antioxidant status may provide an opportunity to enhance repair and improve recovery in patients with injury.

No Evidence of Hematopoietic Stem Cell Mobilization in Patients Submitted to Hepatectomy or in Patients With Acute on Chronic Liver Failure

Background Liver regeneration is a heterogeneous phenomenon involving the proliferation of different cell lineages in response to injury. Under a strong positive selection pressure bone marrow derived stem cells may be involved in this process, by making a contribution to both parenchymal restoration and endothelial cell replacement. We investigate bone marrow stem cell migration to the liver in patients undergoing hepatectomy or with acute on chronic liver failure. Methods We enrolled 6 patients submitted to hepatectomy, 6 patients to cholecystectomy and 8 patients with acute decompensation of liver cirrhosis. Mobilization of CD34+ cells was evaluated by cytofluorimetry on peripheral blood samples at different time points; baseline, 1, 3, 7, 15 and 30 days after surgery and at admission, 1, 7 and discharge among patients with acute on chronic liver failure. 10 healthy subjects undergoing blood donation were also enrolled to evaluated the basal value of CD34+ cells. Results White blood cell counts remained in the normal range (4.1–9.8 × 10 9/L) in all groups throughout the follow-up. In all patients of Groups 1, 2 and 3, circulating CD34+ failed to show statistically significant differences both as the absolute number and as the percentage at any time point compared to healthy controls. Conclusions Bone marrow derived cell mobilization can not be detected after hepatectomy or during an acute decompensation on a cirrhotic liver. Under these circumstances liver regeneration can probably call upon mature hepatocytes and endogenous progenitor cells. The involvement of extrahepatic progenitors if any, is a rare and limited phenomenon.