Dextran sulfate-amplified extracellular matrix deposition promotes osteogenic differentiation of mesenchymal stem cells.

The development of bone-like tissues in vitro that exhibit key features similar to those in vivo is needed to produce tissue models for drug screening and the study of bone physiology and disease pathogenesis. Extracellular matrix (ECM) is a predominant component of bone in vivo; however, as ECM assembly is sub-optimal in vitro, current bone tissue engineering approaches are limited by an imbalance in ECM-to-cell ratio. We amplified the deposition of osteoblastic ECM by supplementing dextran sulfate (DxS) into osteogenically induced cultures of human mesenchymal stem cells (MSCs). DxS, previously implicated to act as a macromolecular crowder, was recently demonstrated to aggregate and co-precipitate major ECM components, including collagen type I, thereby amplifying its deposition. This effect was re-confirmed for MSC cultures undergoing osteogenic induction, where DxS supplementation augmented collagen type I deposition, accompanied by extracellular osteocalcin accumulation. The resulting differentiated osteoblasts exhibited a more mature osteogenic gene expression profile, indicated by a strong upregulation of the intermediate and late osteogenic markers ALP and OCN, respectively. The associated cellular microenvironment was also enriched in bone morphogenetic protein 2 (BMP-2). Interestingly, the resulting decellularized matrices exhibited the strongest osteo-inductive effects on re-seeded MSCs, promoted cell proliferation, osteogenic marker expression and ECM calcification. Taken together, these findings suggest that DxS-mediated enhancement of osteogenic differentiation by MSCs is mediated by the amplified ECM, which is enriched in osteo-inductive factors. We have thus established a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with sequestration of osteo-inductive factors. STATEMENT OF SIGNIFICANCE: As extracellular matrix (ECM) assembly is significantly retarded in vitro, the imbalance in ECM-to-cell ratio hampers current in vitro bone tissue engineering approaches in their ability to faithfully resemble their in vivo counterpart. We addressed this limitation by leveraging a poly-electrolyte mediated co-assembly and amplified deposition of ECM during osteogenic differentiation of human mesenchymal stem cells (MSCs). The resulting pericelluar space in culture was enriched in organic and inorganic bone ECM components, as well as osteo-inductive factors, which promoted the differentiation of MSCs towards a more mature osteoblastic phenotype. These findings thus demonstrated a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with a closer recapitulation of the in vivo tissue niche.

Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription.

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

Methods to analyze the homing efficiency and spatial distribution of hematopoietic stem and progenitor cells and their relationship to the bone marrow endosteum and vascular endothelium.

The tracking of immunofluorescent labeled hematopoietic stem and progenitor cells (HSC/HPC) within the bone marrow (BM) cavity allows the assessment of the regulatory processes involved in transendothelial migration, trans-marrow migration, and finally lodgement into the HSC niche. This is of interest as the extracellular and cellular components involved in the regulation of HSC quiescence and differentiation are still not completely understood. Homing of transplanted HSC is the first critical step in the interaction between HSC and the microenvironment of the BM. As a consequence, murine models allowing the evaluation of the structural relationship between migrating HSC, the endosteal bone surface, and the vascular components of the BM enhance our understanding of hematopoietic regulation.

Developmental fate determination and marker discovery in hematopoietic stem cell biology using proteomic fingerprinting.

In hematopoiesis, co-expression of Sca-1 and c-Kit defines cells (LS(+)K) with long term reconstituting potential. In contrast, poorly characterized LS(-)K cells fail to reconstitute lethally irradiated recipients. Relative quantification mass spectrometry and transcriptional profiling were used to characterize LS(+)K and LS(-)K cells. This approach yielded data on >1200 proteins. Only 32% of protein changes correlated to mRNA modulation demonstrating post-translational protein regulation in early hematopoietic development. LS(+)K cells had lower expression of protein synthesis proteins but did express proteins associated with mature cell function. Major increases in erythroid development proteins were observed in LS(-)K cells; based on this assessment of erythroid potential we showed them to be principally erythroid progenitors, demonstrating effective use of discovery proteomics for definition of primitive cells.

Detection and quantification of functionally defined hematopoietic progenitor cells and tissue specific mRNA within the peripheral blood of myeloma patients after administration of granulocyte colony-stimulating factor and erythropoietin.

OBJECTIVE: Hematopoietic progenitor cells (HPC) as well as tissue committed stem cells expressing mRNA specific to various somatic tissues are thought to be part of the CD34+ bone marrow compartment. In this study, we explore and quantify their mobilization in patients with multiple myeloma undergoing chemotherapy upon administration of granulocyte colony-stimulating factor (G-CSF) plus/minus erythropoietin (EPO). PATIENTS AND METHODS: HPC were quantified by flow cytometry and functional assays within the blood of healthy donors and myeloma patients before and after chemotherapy followed by G-CSF or G-CSF + EPO given subcutaneously. The mRNA expression was studied by quantitative polymerase chain reaction (PCR). Cytokines and peripheral blood protease levels were measured by an enzyme-linked immunosorbent assay. RESULTS: EPO did not significantly alter the number of HPC mobilized by G-CSF alone, and mRNA specific for liver, brain, muscle and kidney was detected in both treatment groups. Quantitative PCR analysis revealed a 2.7-fold increased expression of glial fibrillary acidic protein after G-CSF + EPO administration compared to G-CSF alone (P = 0.003). The concentration of G-CSF rose from 62 +/- 22 pg/mL and 48 +/- 10 pg/mL to 28 +/- 9 ng/mL and 85 +/- 10 ng/mL after 10 d of treatment with G-CSF and G-CSF + EPO, respectively. The concentration of neutrophil elastase (NE) rose only in the G-CSF group by a factor 1.5. CONCLUSION: The alteration of G-CSF and NE levels as well as the expression of tissue committed RNA after the administration of EPO in addition to G-CSF indicate that different growth factors mobilize different stem cells that might potentially be used for the support of tissue repair in future treatment protocols.