Stromal Progenitor Cells Research Articles

Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: a cell source with enhanced commitment to the chondrogenic lineage

Background aimsPrevious studies have demonstrated that porcine synovial membrane stem cells can adhere to a cartilage defect in vivo through the use of a tissue-engineered construct approach. To optimize this model, we wanted to compare effectiveness of tissue sources to determine whether porcine synovial fluid, synovial membrane, bone marrow and skin sources replicate our understanding of synovial fluid mesenchymal stromal cells or mesenchymal progenitor cells from humans both at the population level and the single-cell level. Synovial fluid clones were subsequently isolated and characterized to identify cells with a highly characterized optimal phenotype. MethodsThe chondrogenic, osteogenic and adipogenic potentials were assessed in vitro for skin, bone marrow, adipose, synovial fluid and synovial membrane–derived stem cells. Synovial fluid cells then underwent limiting dilution analysis to isolate single clonal populations. These clonal populations were assessed for proliferative and differentiation potential by use of standardized protocols. ResultsPorcine-derived cells demonstrated the same relationship between cell sources as that demonstrated previously for humans, suggesting that the pig may be an ideal preclinical animal model. Synovial fluid cells demonstrated the highest chondrogenic potential that was further characterized, demonstrating the existence of a unique clonal phenotype with enhanced chondrogenic potential. ConclusionsPorcine stem cells demonstrate characteristics similar to those in human-derived mesenchymal stromal cells from the same sources. Synovial fluid–derived stem cells contain an inherent phenotype that may be optimal for cartilage repair. This must be more fully investigated for future use in the in vivo tissue-engineered construct approach in this physiologically relevant preclinical porcine model.

Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2–/–) Mice

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2(-/-) mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2(-/-) mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2(-/-) mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2(+/+) stromal cells (Fancd2(-/-) D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2(+/+) D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2(-/-) IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2(+/+) (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2(-/-) marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2(-/-) stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2(+/+) cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2(-/-) IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2(-/-) stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.

Lysophosphatidic Acid Enhances Stromal Cell-Directed Angiogenesis

Ischemic diseases such as peripheral vascular disease (PVD) affect more than 15% of the general population and in severe cases result in ulcers, necrosis, and limb loss. While the therapeutic delivery of growth factors to promote angiogenesis has been widely investigated, large-scale implementation is limited by strategies to effectively deliver costly recombinant proteins. Multipotent adipose-derived stromal cells (ASC) and progenitor cells from other tissue compartments secrete bioactive concentrations of angiogenic molecules, making cell-based strategies for in situ delivery of angiogenic cytokines an exciting alternative to the use of recombinant proteins. Here, we show that the phospholipid lysophosphatidic acid (LPA) synergistically improves the proangiogenic effects of ASC in ischemia. We found that LPA upregulates angiogenic growth factor production by ASC under two- and three-dimensional in vitro models of serum deprivation and hypoxia (SD/H), and that these factors significantly enhance endothelial cell migration. The concurrent delivery of LPA and ASC in fibrin gels significantly improves vascularization in a murine critical hindlimb ischemia model compared to LPA or ASC alone, thus exhibiting the translational potential of this method. Furthermore, these results are achieved using an inexpensive lipid molecule, which is orders-of-magnitude less costly than recombinant growth factors that are under investigation for similar use. Our results demonstrate a novel strategy for enhancing cell-based strategies for therapeutic angiogenesis, with significant applications for treating ischemic diseases.

Cancer Risk Is Not Increased in Patients Treated for Orthopaedic Diseases with Autologous Bone Marrow Cell Concentrate

There is concern that regenerative cell-based therapies could result in increased risk of tumor formation. We investigated the long-term risks for systemic and site-specific cancers in patients who had received autologous bone marrow-derived stromal progenitor cells to treat orthopaedic lesions. A total of 1873 patients were treated from 1990 to 2006 with bone marrow-derived concentrated cells. Patients were monitored for cancer incidence from the date of the first operation (1990) until death, or until December 31, 2011. The mean follow-up time was 12.5 years (range, five to twenty-two years). The average number of colony-forming unit fibroblasts returned to the patients was 483,000 fibroblasts (range, 62,000 to 2,095,000 fibroblasts). The primary outcome was to evaluate with radiographs and/or magnetic resonance imaging the risk of tumorigenesis at the cell therapy treatment sites. The secondary outcome was to evaluate the risk of cancer diagnosed in areas other than the treatment site during the follow-up period. The relative risk of cancer was expressed as the ratio of observed and expected number of cases, that is, the standardized incidence ratio, according to the cancer incidence in the French population. No tumor formation was found at the treatment sites on the 7306 magnetic resonance images and 52,430 radiographs among the 1873 patients. Fifty-three cancers were diagnosed in areas other than the treatment site. On the basis of cancer incidence in the general population during the same period, the expected number of cancers was between ninety-seven and 108 for the same age and sex distribution. The range of the standardized incidence ratio for the follow-up period was between 0.49 and 0.54 (95% confidence interval, 0.30 to 0.80). This study found no increased cancer risk in patients after application of autologous cell-based therapy using bone marrow-derived stromal progenitor cells either at the treatment site or elsewhere in the patients after an average follow-up period of 12.5 years.

FGF2 induces RANKL gene expression as well as IL1β regulated MHC class II in human bone marrow-derived mesenchymal progenitor stromal cells

Human bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1β and IFNγ. RANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1β and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR. In hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1β and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1β induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1β through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1β in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts. RANKL expression and IL1β regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1β and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.

Regulation Of Hematopoietic Stem Cell Trafficking By The Coagulation Pathway

Hematopoeitic stem and progenitor cells (HSPC) dynamically switch between a quiescent, non-motile mode in the bone marrow (BM), to an active state, in which they proliferate, differentiate and egress to the circulation. Injection of the coagulation protease thrombin induced rapid HSPC mobilization to the blood via activation of its major receptor, protease activated receptor 1 (PAR1) on BM hematopoietic and stromal cells. We hypothesized that coagulation factors control stem cells fate in the BM. We examined if thrombin is generated in the murine BM and found by immunohistochemistry prothrombin associated with bone lining osteoblasts in the endosteum region. These cells also highly express osteopontin which induces stem cell quiescence and retention. Cleavage of osteopontin by thrombin or by osteoclast derived cathepsin K induces stem cell mobilization. In addition, a unique structure of multinucleated CD45+ cell clusters in the trabecular-rich area of the murine femoral metaphysis express the cell surface receptor Tissue Factor (TF), a potent initiator of the coagulation cascade leading to thrombin generation. These clusters were found adjacent to multinucleated TRAP (tatrate resistant acid phosphate) positive active osteoclasts. In vitro, we found that immature osteoclasts expressed TF in cell fusion areas, suggesting that osteoclast maturation also activates the coagulation thrombin/PAR1 axis, thus mediating HSPC recruitment to the circulation. Supporting this notion, bleeding which prompts a hemostatic response and thrombin production, is a strong inducer of osteoclasts activation and HSPC mobilization. In addition, injection of bacterial lipopolysaccharides (LPS) is known to activate osteoclasts and induce HSPC mobilization (Kollet et al Nat Med 06). We found that LPS injection upregulated TF expression by CD45+ myeloid cells in the murine BM. LPS treatment provoked massive HSPC mobilization, which was attenuated by PAR1 inhibition. To further address the role of thrombin in stem cell maintenance, we targeted prothrombin in vivo by applying Antisense Oligonucleotides (ASO) knockdown technology, previously shown to induce a dose- and time-dependent up to 90% reduction of prothrombin mRNA levels in the murine liver (Monia et al Blood 2010). Prothrombin depletion altered the BM niche microenvironment by expanding the mesenchymal stem and progenitor (MSPC) population and the long-term repopulating CD34-/ROSlow/LSK HSPC population in the BM. In untreated mice, TF was also expressed by a small MSPC population, suggesting that the bone stomal compartment may also contribute to the regulation of HSPC mobilization upon demand. To further asses the role of thrombin generation in HSPC development, we examined the involvement of the endothelial cell receptor Thrombomodulin (TM) that is pivotal for the anticoagulant pathway which mediates activation of protein C. TM protein is expressed by BM small blood vessels resembling sinusoids and by neighboring MSPC. By immunohistochemistry, we also detected activated protein C on the same blood vessels. A mouse model with a mutation in the TM gene (TMPro/Pro) is characterized by reduced capacity for activated protein C generation which in turn increases thrombin levels in these mice. We found increased circulating hematopoietic stem cells in TMPro/Pro mice, suggesting that chronically increased basal levels of thrombin generation can promote HSC egress. Conversely, short term (5 day) intermittent treatment of mice with low dose thrombin that mainly causes activated protein C formation in vivo, display higher levels of CD34-/ROSlow/LSK and EPCR+LSK stem cells in the BM, indicating additional roles for the anticoagulant pathway in BM stem cell pool maintenance. In summary, our results provide evidence that the activator of the coagulation cascade, TF, and coagulation factors Thrombin and activated protein C are present in the BM and regulate and integrate functions of hematopoietic stem and progenitor cells and BM stromal progenitor cells. Disclosures: Crosby: Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other. Monia:Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other.

New Light Is Shed on the Enigmatic Origin of the Lung Myofibroblast

New Light Is Shed on the Enigmatic Origin of the Lung Myofibroblast

Implications of Immediate Autologous Fat Grafting and Expanded Adipose-Derived Progenitor Cells on Hypertrophic Scar Maturation in a Swine Model

INTRODUCTION: Hypertrophic scar formation is unpredictable and poorly understood, afflicting both the pediatric and adult populations. Treatment methods with conservative and invasive approaches have low rates of compliance and high rates of morbidity. The purpose of this study is to develop a reproducible scar model and investigate a new technique of scar modification through the use of adipose-derived progenitor stromal cells (ASCs). METHODS: 20 thermal deep-partial thickness contact burns were created on the dorsum of three 8-week old domestic swine and allowed to mature for 10 weeks. Scars were then injected with 2 cc saline, expanded autologous ASCs, or 2 cc fresh lipoaspirate and sampled at 2 week intervals up to 10 weeks post injection. Volumetric analysis with a 3-D scanner, mechanical elasticity testing through negative pressure transduction, and standardized photography evaluation with Image J was performed. Biopsies were taken at 2 week intervals and histologically analyzed for collagen deposition and vascularity. RNA sequencing was performed on scar tissue samples, cultured cells, and fresh lipoaspirate. RESULTS: Volumetric analysis demonstrates a reduction in average scar thickness at 6 weeks when injected with ASCs (-1.601 cc³) and autologous fat (-1.965 cc³) relative to controls (-0.121 cc³, p<0.05). A decrease in overall tissue compliance is observed with fat or ASC injection when compared to unburned skin at 8 weeks (35.99/ 37.94 vs 49.36 mmHg*mm, p<0.01). Scars treated with fat or ASCs increase the rate at which erythema resolves. RNA sequencing suggests regulation of fibroblast gene expression and a decreased inflammatory profile when scars are injected with fat cells. CONCLUSION: Early results suggest that autologous fat and/or ASCs may improve healing of hypertrophic scarring by altering the cellular and structural components during wound remodeling up to 14 weeks after injury. This may have beneficial applications in early treatment of large or cosmetically sensitive immature burn scars (Figures 1 and 2).Figure 1: 3-D volumetric analysis of hypertrophic scars. Scans at 2 and 4 weeks were superimposed (top left, top middle, top right) and thickness differences (arrow) calculated between time points (bottom).Figure 2: Biochemical evaluation through negative pressure transduction demonstrates a decrease in overall elasticity in all injected wounds (saline, cells, fat) up to 4 weeks. Elasticity increases in each wound starting around week 6.

Three‐dimensional culture of differentiated endometrial stromal cells to oligodendrocyte progenitor cells (OPCs) in fibrin hydrogel

Neural tissue engineering is one of the most promising strategies for treatment of nerve tissue injuries. Three-dimensional (3D) environment mimics in vivo conditions for cells. 3D distribution and growth of the cells within the scaffold are both important for neural tissue engineering. In this study, endometrial stromal cell-derived oligodendrocyte progenitor cells (EnSC-derived OPCs) were cultured in fibrin gel and cell differentiation and viability were evaluated after 8 days of post-culture. The structural and mechanical characteristics of fibrin gel-like scaffold were examined with rheological analysis. EnSCs were isolated from donor tissue and were induced to OPCs with growth factors (FGF2/EGF/PDGF-AA) for 12 days, then were cultured in fibrin gel with Triiodothyronine (T3) medium for another 8 days. The viability of cells was analyzed using MTT assay for a period of 8 days culturing in a fibrin matrix. Structure of fibrin matrix and cell morphology was analyzed with SEM. TEM, immunostaining and quantitative RT-PCR was performed for OPCs markers after cell culturing in fibrin matrix. Cell viability is enhanced in fibrin matrix after 8 days. SEM and TEM show that cells are in good integration with nano-fibers. Moreover, immunohistochemistry and quantitative RT-PCR of OPCs differentiation markers showed that Olig2, Sox10, PDGFRa, CNP, and A2B5 are expressed after 8 days culturing within fibrin matrix. Fibrin can provide a suitable 3-D scaffold for EnSCs differentiated cells for the regeneration of CNS.

The effect of electrically charged polyion complex nanoparticle-coated surfaces on adipose-derived stromal progenitor cell behaviour

Surface characteristics of biomaterials such as wettability, rigidity, roughness, and electrical charge affect the fate of transplanted cells such as progenitor cells or stem cells for use in regenerative medicine. Of these, the effects of surface electrical charges on cellular behaviour such as adhesion, proliferation, and differentiation are not well understood. We prepared precisely charged culture surfaces ranging from −28 mV to +21 mV, simply by surface deposition of polyion complex nanoparticles prepared by mixing a positively charged thermoresponsive homopolymer, poly(N,N-dimethylaminoethyl methacrylate), with negatively charged plasmid DNA at various charge ratios. Drastic morphological changes of adipose-derived vascular progenitor cells were generated on the positively charged surface of organized forms at +19 mV. Capillary-like networks or single aggregates of these cells were selectively created depending on cell seeding density. Our findings offer new insights that may aid develop stem cell-processing techniques for use in regenerative medicine.

Transplantation of Bone Marrow Stromal Cell-Derived Neural Precursor Cells Ameliorates Deficits in a Rat Model of Complete Spinal Cord Transection

After severe spinal cord injury, spontaneous functional recovery is limited. Numerous studies have demonstrated cell transplantation as a reliable therapeutic approach. However, it remains unknown whether grafted neuronal cells could replace lost neurons and reconstruct neuronal networks in the injured spinal cord. To address this issue, we transplanted bone marrow stromal cell-derived neural progenitor cells (BM-NPCs) in a rat model of complete spinal cord transection 9 days after the injury. BM-NPCs were induced from bone marrow stromal cells (BMSCs) by gene transfer of the Notch-1 intracellular domain followed by culturing in the neurosphere method. As reported previously, BM-NPCs differentiated into neuronal cells in a highly selective manner in vitro. We assessed hind limb movements of the animals weekly for 7 weeks to monitor functional recovery after local injection of BM-NPCs to the transected site. To test the sensory recovery, we performed functional magnetic resonance imaging (fMRI) using electrical stimulation of the hind limbs. In the injured spinal cord, transplanted BM-NPCs were confirmed to express neuronal markers 7 weeks following the transplantation. Grafted cells successfully extended neurites beyond the transected portion of the spinal cord. Adjacent localization of synaptophysin and PSD-95 in the transplanted cells suggested synaptic formations. These results indicated survival and successful differentiation of BM-NPCs in the severely injured spinal cord. Importantly, rats that received BM-NPCs demonstrated significant motor recovery when compared to the vehicle injection group. Volumes of the fMRI signals in somatosensory cortex were larger in the BM-NPC-grafted animals. However, neuronal activity was diverse and not confined to the original hind limb territory in the somatosensory cortex. Therefore, reconstruction of neuronal networks was not clearly confirmed. Our results indicated BM-NPCs as an effective method to deliver neuronal lineage cells in a severely injured spinal cord. However, reestablishment of neuronal networks in completed transected spinal cord was still a challenging task.

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model

Background aimsDelivery of bone marrow–derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing. MethodsA transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro–computed tomography. ResultsAMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84. ConclusionsOur data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.

Extraprostatic Extension into Periprostatic Fat is a More Important Determinant of Prostate Cancer Recurrence than an Invasive Phenotype

Although micrometastasis development correlates closely with the depth of invasion of many tumor types, it is unclear whether invasion into but not through the prostatic pseudocapsule has a negative impact on prognosis, similar to extraprostatic extension. We defined the impact of pseudocapsular invasion on the risk of post-prostatectomy biochemical recurrence. Patients with pT2-3a prostate cancer were identified from a prospectively recorded database. Those with pT2 disease were categorized according to pseudocapsular invasion presence or absence. The impact of pseudocapsular invasion on biochemical recurrence was determined by univariable and multivariable Cox regression analysis. In a cohort of 1,338 patients we identified 595 with organ confined cancer positive for pseudocapsular invasion. Compared to tumors without evidence of invasion, pseudocapsular invasion was positively associated with higher Gleason grade and tumor volume (1.2 vs 1.9 cc, each p<0.001). On univariable analysis there was no difference in biochemical recurrence-free survival between patients with vs without pseudocapsular invasion, although those with extraprostatic extension had significantly lower biochemical recurrence-free survival (p<0.001). This was confirmed on multivariable analysis, which revealed that extraprostatic extension was a significant independent predictor of biochemical recurrence (HR 1.53, p=0.018). The presence of pseudocapsular invasion had no effect (HR 0.81, p=0.33). Pseudocapsular invasion is not a pathological feature associated with an adverse outcome after prostatectomy. Thus, the depth of tumor invasion is not a continuum of risk and access to periprostatic adipose tissue is a more important determinant of disease behavior than an invasive phenotype.

Physiologic corticosterone oscillations regulate murine hematopoietic stem/progenitor cell proliferation and CXCL12 expression by bone marrow stromal progenitors

The role of corticosterone (Cort), the immune system's major stress hormone, in the regulation of hematopoietic stem and progenitor cells (HSPCs) and their dynamic bone marrow (BM) microenvironment is currently unknown. We report that corticotropin-releasing factor receptor 1 (CRFR1) mutant mice with chronically low Cort levels showed aberrant HSPC regulation, having higher HSPC numbers and upregulation of the chemokine CXCL12, phenotypes that were restored by Cort supplementation. Expanded stromal progenitors known to support HSPCs were also observed in these low-Cort-containing mice. A similar phenotype was induced in wild-type (WT) mice by Metyrapone, a Cort synthesis inhibitor. Conversely, high Cort exposure induced HSPC apoptosis, reduced long-term BM repopulation and decreased stromal progenitor cell numbers. We documented circadian oscillations of Cort in WT BM but not in CRFR1 mutant mice, leading to diminished circadian BM CXCL12 fluctuations and increased number of circulating HSPCs in these mice. Finally, low Cort induced expansion of stromal progenitors, CXCL12 expression, HSPC proliferation and BM repopulation capacity, involving Notch1 signaling. This was associated with upregulation of the Notch ligand, Jagged1, in BM myeloid cells. Our results suggest that daily physiologic Cort oscillations are critical for balanced HSPC proliferation and function involving Notch1 signaling and their supportive BM microenvironment.

P-254 Chromosomal analysis of hematopoietic and stromal progenitor cells in patients with myelodysplastic syndrome (MDS)

P-254 Chromosomal analysis of hematopoietic and stromal progenitor cells in patients with myelodysplastic syndrome (MDS)

Priming with secreted ligands from human stromal progenitor cells (p75MSCs) promotes grafts of adult cardiac stem/progenitor cells after myocardial infarction

Priming with secreted ligands from human stromal progenitor cells (p75MSCs) promotes grafts of adult cardiac stem/progenitor cells after myocardial infarction

Mobilization of progenitor cells into peripheral blood by gamma-tocotrienol: A promising radiation countermeasure

Gamma-tocotrienol (GT3), a vitamin E isoform, is shown to induce high levels of granulocyte colony stimulating factor (G-CSF) in mice. G-CSF is a key cytokine used for stimulation of hematopoiesis, and mobilization of hematopoietic stem and progenitor cells into peripheral blood. GT3 is also shown to induce vascular endothelial growth factor (VEGF), another important cytokine necessary for vasculogenesis and endothelial progenitor mobilization. Since GT3 induces both these cytokines, we tested whether GT3 mobilizes hematopoietic and endothelial progenitors in mice. GT3 (200mg/kg) was injected in 10-week-old CD2F1 mice and mobilization of progenitors in peripheral blood was analyzed at 24, 48, and 72h post-administration. Circulating hematopoietic progenitor cells (HPCs, Lin−, cKit+), endothelial progenitor cells (EPCs, Lin−, CD34+, Flk+), and stromal progenitor cells (SPCs, Lin−, CD29+, CD105+) in peripheral blood mononuclear cells (PBMCs) were analyzed simultaneously by flow cytometry. Mobilized HPCs, EPCs and SPCs in PBMC were also measured by colony-forming unit (CFU) assay in progenitor-specific media. Three groups of mice received vehicle, GT3 and GT3 plus AMD3100, a receptor antagonist used to enhance mobilization. GT3 induced significant mobilization of all three progenitor cell types compared to vehicle in peripheral blood; AMD3100 enhanced GT3-induced mobilization even further. Mobilization of progenitor cells in peripheral blood by GT3 indicates that GT3 can be used as an alternative to G-CSF and VGEF to mobilize HPCs and EPCs.

RNA Trafficking by Acute Myelogenous Leukemia Exosomes

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML.

Mesenchymal Stem Cell: Keystone of the Hematopoietic Stem Cell Niche and a Stepping-Stone for Regenerative Medicine

Mesenchymal stem cells (MSCs) are self-renewing precursor cells that can differentiate into bone, fat, cartilage, and stromal cells of the bone marrow. Recent studies suggest that MSCs themselves are critical for forming a niche that maintains hematopoietic stem cells (HSCs). The ease by which human MSC-like and stromal progenitor cells can be isolated from the bone marrow and other tissues has led to the rapid development of clinical investigations exploring their anti-inflammatory properties, tissue preservation capabilities, and regenerative potential. However, the identity of genuine MSCs and their specific contributions to these various beneficial effects have remained enigmatic. In this article, we examine the definition of MSCs and discuss the importance of rigorously characterizing their stem cell activity. We review their role and that of other putative niche constituents in the regulation of bone marrow HSCs. Additionally, how MSCs and their stromal progeny alter immune function is discussed, as well as potential therapeutic implications.

CXCL12/CXCR4 signal axis plays an important role in mediating bone morphogenetic protein 9-induced osteogenic differentiation of mesenchymal stem cells.

Mesenchymal progenitor stem cells (MPCs) are a group of bone marrow stromal progenitor cells processing osteogenic, chondrogenic, adipogenic and myogenic lineages differentiations. Previous studies have demonstrated that bone morphogeneic protein 9(BMP9) is one of the most osteogenic BMPs both in vitro and in vivo, however, the underlying molecular mechanism of osteogenesis induced by BMP9 is needed to be deep explored. Here, we used the recombinant adenoviruses assay to introduce BMP9 into C3H10T1/2 mesenchymal stem cells to elucidate the role of CXCL12/CXCR4 signal axis during BMP9-incuced osteogenic differentiation. The results showed that CXCL12 and CXCR4 expressions were down-regulated at the stage of BMP9-induced osteogenic differentiation, in a dose- and time-dependent. Pretreatment of C3H10T1/2 cells with CXCL12/CXCR4 could significantly affect the early and mid osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN), the transcription factors of Runx2, Osx, Plzf and Dlx5 expression, through activating the Smad, MAPK signaling pathway. Addition of exogenous CXCL12 did not affect the changes of the late osteogenic marker calcium deposition. Thus, our findings suggest a co-requirement of the CXCL12/CXCR4 signal axis in BMP9-induced the early- and mid-process of osteogenic differentiation of MSCs.