BM-derived Stem Cells Research Articles

Abstract WP99: Post-stroke PPARγ Activation Promotes The Proliferation And Differentiation Of Innate- And Bone Marrow-derived Stem Cells In Subventicular Zone, Leading To Vasogenesis And Neurogenesis In Cortex

Background and Purpose— Neuroprotection after ischemia by pretreatment with a peroxisome proliferator-activated receptor gamma (PPARγ) agonist was associated with the transcriptional activation of anti-apoptotic- and survival genes via p-STAT3 in oophorectomized (OVX) rats. Based on this finding, we hypothesized that post-stroke PPARγ activation contributes to neurogenesis. Methods— To examine the proliferation and differentiation after cerebral ischemia of innate- and bone marrow (BM)-derived stem cells, we treated 13 week-old OVX Wistar rats with the PPARγ agonist. After replacing their BM cells with BM cells from green fluorescent protein transgenic (GFP) rats they were subjected to 90-min cerebral ischemia and divided into 2 groups. One was treated with pioglitazone (PGZ, 2.5 mg/kg/day) for 14 days after ischemia induction. The other was vehicle-control (VC). Results— Compared to VC, neurological deficits and infarct volumes were significantly reduced in rats treated with PGZ. In the VC rats innate- and BM-derived stem cells in the subventricular zone (SVZ) were slightly increased after ischemia. Notably, in the PGZ group, the proliferation of both cell types was evidently augmented with PPARγ expression. They were translocated from the SVZ into the cortical peri-infarct area. There they differentiated into neurons, glia, and blood vessels and the expression of phosphorylated Akt, VEGF, and of BrdU incorporated into the cells was increased. This suggested the PGZ-induced vasogenesis and neurogenesis via activation of cell survival- and proliferation signals, resulting in the neurological improvement and the reduction of infarct size. Conclusions— Our findings provide new evidence in OVX rats that PPARγ activation after ischemia promotes the proliferation and differentiation of innate- and BM stem cells, leading to good outcome.

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

Cell Therapy for Diabetic Neuropathy Using Adult Stem or Progenitor Cells

Diabetic neuropathy (DN) is the most common and disabling complication of diabetes that may lead to foot ulcers and limb amputations. Despite widespread awareness of DN, the only effective treatments are glucose control and pain management. A growing body of evidence suggests that DN is characterized by reduction of vascularity in peripheral nerves and deficiency in neurotrophic and angiogenic factors. Previous studies have tried to introduce neurotrophic or angiogenic factors in the form of protein or gene for therapy, but the effect was not significant. Recent studies have shown that bone marrow (BM)-derived stem or progenitor cells have favorable effects on the repair of cardiovascular diseases. Since these BM-derived stem or progenitor cells contain various angiogenic and neurotrophic factors, these cells have been attempted for treating experimental DN, and turned out to be effective for reversing various manifestations of experimental DN. These evidences suggest that cell therapy, affecting both vascular and neural components, can represent a novel therapeutic option for treatment of clinical DN.

Novel Role for Bioactive Lipids in Mobilization of Bone Marrow Stem Cells During Myocardial Ischemia: Sphingosine-1 Phosphate (S1P) As Potential Therapeutic Target

Abstract 1911 Background:Bone marrow (BM) contains a variety of stem cells, including mobile pool of hematopoietic stem cells and non-hematopoietic stem cells and acute myocardial infarction (AMI) triggers mobilization of BM-derived stem cells (BMSCs) through poorly understood processes. Recently we have postulated a major role for bioactive lipids such as sphingosine-1 phosphate (S1P) in G-CSF- and AMD3100-induced mobilization of hematopoietic stem cells (HSCs) into peripheral blood (PB) (Leukemia 2010;24:976–85). Hypothesis:We hypothesized that S1P could also play a role in mobilization of BMSCs in patients during tissue and organ injury as seen for example in patients with AMI. Methods:Peripheral blood (PB) samples from matched controls and patients presenting with ST-elevation myocardial infarction (STEMI) were examined i) by employing FACS for a number of circulating in PB lineage negative (Lin-)/CD45-/CD34+ and CD133+ and CXCR4+ BMSCs, ii) PB level of S1P by employing mass spectrometry, iii) SDF-1 level by ELISA and finally iv) BMSCs were tested in Transwell migration assays for their chemotactic responsiveness to S1P gradient in plasma from STEMI patients. Results:Plasma S1P levels were highest within 6 hours of AMI presentation 0.31 ± 0.02 μM and declined to 0.14 ± 0.02 μM in controls (P < 0.05 for 6 hours vs. controls). The elevation of plasma S1P corresponded with complement cascade (CC) activation and higher levels of C5b-C9 membrane attack complex (MAC) complex in the plasma. The largest reservoir of S1P in whole blood was red blood cells (RBCs) and incubation of RBCs with activated complement initiated the release of S1P. Elevated plasma S1P levels was paralleled by early significant increase in circulating lineage negative (Lin-)/CD45-/CD34+, CD133+ and CXCR4+ BMSCs (P < 0.01 vs. controls). Plasma obtained from STEMI patients in the early phases following acute injury stimulated the migration of human BM-derived Lin-/CXCR4+ and Lin-/CD34+ BMSCs in chemotaxis assays (7–8-fold increase vs. vehicle, P < 0.05), this effect was blunted by charcoal stripping of the plasma (CSP) and was further inhibited by the specific S1P receptor type 1 (S1P1) antagonists W146 (10 μM) and VPC20139 (10 μM). At the same time to our surprise we did not observed significant chemotactic relevant changes in SDF-1 level in PB plasma. Conclusion:This is the first human study to suggest that elevated S1P level in PB early in the course of AMI mobilizes BMSCs. Furthermore, we demonstrate for the first time that the release of hematopoietic and non-hematopoietic BMSCs into PB correlates in AMI patients with activation of CC and generation of MAC, that may release S1P from erythrocytes and platelets. We postulate that based on these results future studies examining the therapeutic manipulation of S1P and its receptors for myocardial regeneration are warranted. Disclosures:No relevant conflicts of interest to declare.

Induction of Thymic Stromal Lymphopoietin in Mesenchymal Stem Cells by Interaction with Myeloma Cells

AbstractAbstract 1825Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal proliferation of malignant plasma cells in the bone marrow (BM). The development of new drugs has increased survival time, but the disease is still incurable. Recently, the BM microenvironment has been reported to play an important role in proliferation and survival of MM cells, suggesting that clarification of the interaction between BM stromal cells and MM cells may provide insights to allow the development of novel treatment strategies for MM.As the signal between MM cells and the BM microenvironment should be bidirectional, it is possible that MM cells produce signals that alter the BM microenvironment to one appropriate for their survival. To validate this hypothesis, we examined the changes in gene expression profile in human BM-derived mesenchymal stem cells (MSC) by co-culture with MM cell line OPM-2. cDNA array analysis indicated that 2 days of co-culture with OPM-2 induced multiple factors in MSC, which are known to have positive effects on survival of MM cells, such as interleukin-6, insulin-like growth factor-1, stromal cell-derived factor 1, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Other than known factors, thymic stromal lymphopoietin (TSLP), which is a cytokine involved in progression of pancreatic and breast cancer through induction of T helper 2 cell responses, was found to be upregulated by co-culture (ratio 2.22). To confirm the results of cDNA array analysis, MSC co-cultured with OPM-2 or KMS-34 were tested for expression and secretion of TSLP by real-time PCR and ELISA. Real-time PCR revealed that the expression of TSLP in co-cultured MSC was two to three times higher than that in MSC monocultures. Significantly elevated levels of TSLP secretion from MSC after stimulation with PMA/ionomycin were observed by co-culture with OPM-2 or KMS-34 compared with monoculture (201.3 ± 5.0 pg/ml, 176.1 ± 13.3 pg/ml, and 96.6 ± 5.5 pg/ml, respectively; P < 0.01) as measured by ELISA. TSLP did not exhibit any effect on proliferation or drug resistance of MM cells independent of the level of TSLP receptor expression, therefore we evaluated the immunomodulatory effects of TSLP secreted by MSC. Monocyte-derived dendritic cells (MDDC) were first treated with the supernatant of either MSC or MSC co-cultured with KMS-34. Then, allogeneic naïve CD4+ T cells were co-cultured with pretreated MDDC. MDDC treated with the supernatant of co-cultured MSCs expanded IL-13+ CD4+ T cells to a greater extent than MDDC treated with the supernatant of MSC monocultures (10.3 ± 3.7 %, 5.9 ± 4.2 %, respectively; P = 0.02), and addition of anti-TSLP neutralizing antibodies to the supernatant decreased the expansion of IL-13+ CD4+ T cells (10.3 ± 3.7 %, 7.1 ± 2.6 %, respectively; P = 0.04).These results suggested that MM cells act to form more advantageous BM microenvironment for their survival through induction of pleiotropic molecules in MSC involved in not only proliferation and survival of MM cells, but also modifying immunological status. Disclosures:No relevant conflicts of interest to declare.

Novel Evidence That a Quiescent Murine Population of Bone Marrow (BM)-Residing, Developmentally Early, Very Small Sca-1+Lin–CD45– Cells Is Highly Responsive to Prolonged Bleeding by in Vivo Proliferation and Differentiation Into CD45+ Hematopoietic Stem/Progenitor Cells (HSPCs)

Abstract 1249 Background.Several phenotypes have been proposed for long-term repopulating hematopoietic stem cells (LT-HSCs) in murine bone marrow (BM). However, evidence from our and other laboratories has accumulated that adult murine tissues contain a population of developmentally early, so-called very small embryonic-like stem cells (VSELs), which we have proposed as playing an important role as precursors of LT-HSCs (Exp. Hematology 2011;39:225, Leukemia 2012;25,1278). As we reported, these cells are kept quiescent in the BM microenvironment by erasure of the somatic imprint in differentially methylated regions (DMRs) of some developmentally crucial, paternally imprinted genes (Igf2-H19, RasGRF1, and p57Kip2), which proper expression is required for proliferation and expansion of pluripotent stem cells (e.g., embryonic stem cells) (Leukemia 2009;23:2042). However, we also demonstrated that these cells may be specified into the hematopoietic lineage in vitro in co-cultures over OP9 stromal cells. Hypothesis.We hypothesized that these very small cells, which can be specified into the hematopoietic lineage ex vivo in an “artificial” OP9 microenvironment, should also be able to become specified into HSPCs in vivo in the normal BM microenvironment in situations of hematopoietic stress that promote the formation of new HSPCs. Experimental strategies.Normal C57Bl6 mice were bled (twice a week, 200 μl/bleeding) for 4 weeks, and by the end of each week were injected with bromodeoxyuridine (BrdU) to label cells that are in the cell cycle. These mice were subsequently sacrificed and BM cells, flushed from BM cavities as well as from crushed/collagenase-treated bones to recover cells associated with endosteal niches, were obtained from both control and bled mice. In these cell suspensions, we measured i) the total number of Sca-1+Lin–CD45+ HSPCs and small Sca-1+Lin–CD45– VSELs by FACS and ii) the number of cycling BrdU+ HSPCs and VSELs. Moreover, by employing RQ-PCR, we measured the expression of genes regulating the early stages of hematopoiesis and imprinted genes that keep VSELs quiescent in the cell cycle. We also tested the ability of VSELs from control and bled mice to differentiate into CD45+ HSPCs in OP9 co-cultures and their ability to reconstitute hematopoiesis in lethally irradiated mice. Salient results.We observed that the number of cycling BrdU+ VSELs increased from ∼1 ± 0.03% (control) to ∼26 ± 4% and ∼32 ± 6% among BM cells derived from flushed and crushed bones, respectively. Furthermore, in comparison with control animals, BM VSELs isolated from mice after chronic bleeding expressed lower levels of pluripotency markers such as Oct-4 and Nanog, upregulated expression of pro-proliferative mRNA whose expression is regulated by paternal imprinting (Igf2, IGF-1R, and RasGRF1), and downregulated expression of mRNA for paternally imprinted, proliferation-inhibiting H19 and p57Kip2genes. At the same time, the number of BM HSPCs increased from ∼17 ± 3% to 35 ± 7% and 1 ± 0.02% to 40 ± 5% in flushed and crushed bone-derived cells, respectively. Most importantly, we observed that VSELs isolated from bled mice highly upregulated the expression of genes involved in early stages of hematopoiesis, including Ikaros, Lmo2, GATA-2, HoxB4, PU.1, Scl and c-myb, and this correlated with their accelerated ability to become specified into CD45+ HSPCs in co-cultures over OP9 stroma. Finally, VSEL-derived CD45+ HSPCs, when isolated from OP9 cultures, grew methylocelulose colonies from all major hematopoietic lineages and were able to reconstitute hematopoiesis in lethally irradiated recipients. Conclusions.Our data, obtained in an in vivo murine model of hematopoietic stress from chronic bleeding, strongly support the notion that developmentally early murine Sca-1+Lin–CD45– VSELs represent a population of quiescent stem cells in BM that become specified into the hematopoietic lineage in vivo. We propose that, in order to establish the relationship of these cells to other LT-HSC phenotypes described in BM as well as to construct a complete developmental hierarchy, their hematopoietic potential should be compared side-by-side with other BM-derived stem cells isolated using different phenotypic criteria. Disclosures:Ratajczak:Neostem Inc: Member of SAB Other.

CD34+ Hematopoietic Stem Cells Mobilization, Paralleled with Multiple Cytokines Elevated in Patients with HBV-Related Acute-on-Chronic Liver Failure

Recent studies indicate that bone marrow (BM)-derived stem cells contribute to liver regeneration. But limited information is available on the dynamic and mechanisms of mobilization of BM-derived hematopoietic stem cells (HSCs) after acute-on-chronic liver failure (ACLF). The purpose of this study was to assess the mobilization of BM-derived CD34+ HSCs in ACLF patients, and elucidate the association of stress-induced cytokines in HSCs mobilization and/or liver repair in ACLF patients. Thirty patients with HBV-related ACLF, 30 patients undergoing chronic hepatitis B, and 20 healthy controls were enrolled. The percentages of peripheral blood CD34+ cells were determined by two-color flow cytometry. The hepatic commitment of mobilized CD34+ cells was investigated by RT-PCR. The serum levels of stress-induced cytokines were determined by enzyme-linked immunosorbent assays. A significant increase of circulating CD34+ cells was observed in ACLF patients. RT-PCR analyses showed that the mobilized CD34+ cells expressed both CD34 mRNA and liver-specific markers including cytokeratin 19 and α-fetoprotein. In parallel with mobilization of BM-derived CD34+ cells, elevated serum levels of hepatocyte growth factor, interleukin-6, stem cell factor, granulocyte colony-stimulating factor and matrix metalloproteinase 9 were observed in ACLF patients. We demonstrated that ACLF led to mobilization of CD34+ cells, which had a hepatic differentiation potential.

Bone marrow-derived cells can acquire renal stem cells properties and ameliorate ischemia-reperfusion induced acute renal injury

BackgroundBone marrow (BM) stem cells have been reported to contribute to tissue repair after kidney injury model. However, there is no direct evidence so far that BM cells can trans-differentiate into renal stem cells.MethodsTo investigate whether BM stem cells contribute to repopulate the renal stem cell pool, we transplanted BM cells from transgenic mice, expressing enhanced green fluorescent protein (EGFP) into wild-type irradiated recipients. Following hematological reconstitution and ischemia-reperfusion (I/R), Sca-1 and c-Kit positive renal stem cells in kidney were evaluated by immunostaining and flow cytometry analysis. Moreover, granulocyte colony stimulating factor (G-CSF) was administrated to further explore if G-CSF can mobilize BM cells and enhance trans-differentiation efficiency of BM cells into renal stem cells.ResultsBM-derived cells can contribute to the Sca-1+ or c-Kit+ renal progenitor cells population, although most renal stem cells came from indigenous cells. Furthermore, G-CSF administration nearly doubled the frequency of Sca-1+ BM-derived renal stem cells and increased capillary density of I/R injured kidneys.ConclusionsThese findings indicate that BM derived stem cells can give rise to cells that share properties of renal resident stem cell. Moreover, G-CSF mobilization can enhance this effect.

Ischemia/Reperfusion Injury Promotes and Granulocyte-Colony Stimulating Factor Inhibits Migration of Bone Marrow-Derived Stem Cells to Endometrium

The endometrium is a dynamic tissue that undergoes repeated rounds of regeneration in each reproductive (estrous or menstrual) cycle. We have previously shown that bone marrow (BM)-derived stem cells engraft the endometrium in rodents and humans; however, it is not known if these cells contribute physiologically to uterine cyclic regeneration or alternatively are primarily involved in uterine repair in response to injury. Here we performed male-to-female BM transplant and tested the ability of uterine injury to recruit BM-derived cells to endometrium in the presence and absence of sex steroids. Uterine ischemia/reperfusion injury resulted in an ~2-fold increase in BM-derived stem cell recruitment to the endometrium. The effect was independent of sex steroids or the existence of an estrous cycle. BM-derived mesenchymal stem cells (MSCs) are involved in uterine repair after injury, but not the cyclic regeneration of the endometrium in the estrous/menstrual cycle. Granulocyte-colony stimulating factor (G-CSF) is used to increase BM mobilization for transplant and has been proposed as a means of mobilizing stem cells to the uterus. Here G-CSF treatment led to decreased BM engraftment of the uterus after injury, likely by favoring mobilization of hematopoietic stem cells over the MSCs. G-CSF is unlikely to be of benefit in repair of uterine injury in humans. Taken together, we demonstrate that ischemic injury drives BM MSC engraftment of the uterus, independent of estrous cycle, sex steroids, or G-CSF.

Abstract 197: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction

Myocardial infarction (MI) is the primary cause of death throughout the United States. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms may offer novel approaches for G-CSF-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter determined by echocardiogram in WT mice. No improvement on heart function is observed in Plg -/- mice, indicating Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to the ischemic area, BM transplantation with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP + cKit + cells (by 12 fold) in WT mice but not in Plg -/- mice. In addition, BM stem cell-derived vessels and arteries are infrequent in Plg -/- mice suggesting that Plg enhances cardiac repair by promoting stem cell recruitment to the lesion. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in the infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg -/- mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.

Regulation of HIF-1α signaling and chemoresistance in acute lymphocytic leukemia under hypoxic conditions of the bone marrow microenvironment

Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment.

Soluble CCL5 Derived from Bone Marrow-Derived Mesenchymal Stem Cells and Activated by Amyloid β Ameliorates Alzheimer's Disease in Mice by Recruiting Bone Marrow-Induced Microglia Immune Responses

Microglia have the ability to eliminate amyloid β (Aβ) by a cell-specific phagocytic mechanism, and bone marrow (BM) stem cells have shown a beneficial effect through endogenous microglia activation in the brains of Alzheimer's disease (AD) mice. However, the mechanisms underlying BM-induced activation of microglia have not been resolved. Here we show that BM-derived mesenchymal stem cells (MSCs) induced the migration of microglia when exposed to Aβ in vitro. Cytokine array analysis of the BM-MSC media obtained after stimulation by Aβ further revealed elevated release of the chemoattractive factor, CCL5. We also observed that CCL5 was increased when BM-MSCs were transplanted into the brains of Aβ-deposited AD mice, but not normal mice. Interestingly, alternative activation of microglia in AD mice was associated with elevated CCL5 expression following intracerebral BM-MSC transplantation. Furthermore, by generating an AD-green fluorescent protein chimeric mouse, we ascertained that endogenous BM cells, recruited into the brain by CCL5, induced microglial activation. Additionally, we observed that neprilysin and interleukin-4 derived from the alternative microglia were associated with a reduction in Aβ deposition and memory impairment in AD mice. These results suggest that the beneficial effects observed in AD mice after intracerebral SC transplantation may be explained by alternative microglia activation. The recruitment of the alternative microglia into the brain is driven by CCL5 secretion from the transplanted BM-MSCs, which itself is induced by Aβ deposition in the AD brain.

119 Size-dependent retention of stem cells following intracoronary injection

BackgroundIntracoronary (IC) injection of bone marrow mononuclear cells (BMMNCs) is a promising treatment for heart failure. However, therapeutic effects in clinical studies have been small and a principle reason may...

Abstract 13: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction

Ischemic heart disease, including myocardial infarction (MI), is the primary cause of death throughout the US. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms regulating G-CSF-induced cardiac repair may offer novel approaches for strengthening stem cell-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac tissue repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter by echocardiogram in wild-type mice. No improvement in tissue repair and heart function by G-CSF is observed in Plg -/- mice, indicating that Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to ischemia area, bone marrow transplantion (BMT) with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP+ cells (by 16 fold) in WT mice but not in Plg -/- mice, suggesting that Plg is essential for HPSC recruitment from BM to the lesion sites after MI. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg -/- mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.

Recovery From Radiation-induced Bone Marrow Damage by HSP25 Through Tie2 Signaling

Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.

Production of monocytic cells from bone marrow stem cells: therapeutic usage in Alzheimer’s disease

Accumulation of amyloid β (Aβ) is a major hallmark in Alzheimer’s disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aβ burden in mouse models of AD, alleviating the AD pathology. BMM have been shown to be more efficient phagocytes in AD than the endogenous brain microglia. Because BMM have a natural tendency to infiltrate into the injured area, they could be regarded as optimal candidates for cell-based therapy in AD. In this study, we describe a method to obtain monocytic cells from BM-derived haematopoietic stem cells (HSC). Mouse or human HSC were isolated and differentiated in the presence of macrophage colony stimulating factor (MCSF). The cells were characterized by assessing the expression profile of monocyte markers and cytokine response to inflammatory stimulus. The phagocytic capacity was determined with Aβ uptake assay in vitro and Aβ degradation assay of natively formed Aβ deposits ex vivo and in a transgenic APdE9 mouse model of AD in vivo. HSC were lentivirally transduced with enhanced green fluorescent protein (eGFP) to determine the effect of gene modification on the potential of HSC-derived cells for therapeutic purposes. HSC-derived monocytic cells (HSCM) displayed inflammatory responses comparable to microglia and peripheral monocytes. We also show that HSCM contributed to Aβ reduction and could be genetically modified without compromising their function. These monocytic cells could be obtained from human BM or mobilized peripheral blood HSC, indicating a potential therapeutic relevance for AD.

Early sensing of Yersinia pestis airway infection by bone marrow cells

Bacterial infection of the lungs triggers a swift innate immune response that involves the production of cytokines and chemokines that promote recruitment of immune cells from the bone marrow (BM) into the infected tissue and limit the ability of the pathogen to replicate. Recent in vivo studies of pneumonic plague in animal models indicate that the pulmonary pro-inflammatory response to airway infection with Yersinia pestis is substantially delayed in comparison to other pathogens. Consequently, uncontrolled proliferation of the pathogen in the lungs is observed, followed by dissemination to internal organs and death. While the lack of an adequate early immune response in the lung is well described, the response of BM-derived cells is poorly understood. In this study, we show that intranasal (i.n.) infection of mice with a fully virulent Y. pestis strain is sensed early by the BM compartment, resulting in a reduction in CXCR4 levels on BM neutrophils and their subsequent release into the blood 12 hours (h) post infection. In addition, increased levels of BM-derived hematopoietic stem and progenitor cells (HSPC) were detected in the blood early after infection. Mobilization of both immature and mature cells was accompanied by the reduction of BM SDF-1 (CXCL-12) levels and the reciprocal elevation of SDF-1 in the blood 24 h post infection. RT-PCR analysis of RNA collected from total BM cells revealed an early induction of myeloid-associated genes, suggesting a prompt commitment to myeloid lineage differentiation. These findings indicate that lung infection by Y. pestis is sensed by BM cells early after infection, although bacterial colonization of the BM occurs at late disease stages, and point on a potential cross-talk between the lung and the BM at early stages of pneumonic plague.

Leukemic spleen cells are more potent than bone marrow-derived cells in a transgenic mouse model of CML

Spleen size ranks among the most important risk factors in chronic myeloid leukemia (CML), but the pathogenic mechanisms of splenic hematopoiesis in CML remain poorly defined. Here, we studied the biology of Bcr-Abl positive leukemia-initiating cells in the spleen, using an inducible transgenic mouse model of CML. Disease kinetics showed greater increases of immature leukemic cells in spleen vs bone marrow (BM). To assess how Bcr-Abl alters the behavior of spleen-derived CML cells, we transplanted these cells either before ('pre-uninduced') or 44 days after ('pre-induced') expression of the oncogene. Mice transplanted with pre-induced spleen cells showed significantly increased neutrophilia and splenomegaly compared with mice receiving pre-uninduced spleen cells, suggesting that Bcr-Abl expression in the donors had increased splenic tumor burden. However, pre-induction also altered the biology of these cells, as shown by a striking increase in erythropoietic potential. These results differ from those of BM-derived CML stem cells where pre-induction of Bcr-Abl had previously been shown to decrease disease transplantability. Moreover, splenic cells were less sensitive to imatinib than BM cells. In conclusion, Bcr-Abl alters the biology of splenic leukemic stem cells by a cell-autonomous mechanism, but the disease phenotype is also influenced by the microenvironment of these cells.

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy

Transplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model. ASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10(5) cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed. ASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group. Compared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.