Pre-clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice.

BACKGROUND: Platelet (PLT) transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for PLT transfusion products is compounded by a limited supply due to dependency on volunteer donors, a short shelf-life, risk of contaminating pathogens, and alloimmunization. This study provides preclinical evidence that a third-party, cryopreservable source of PLT-generating cells has the potential to complement presently available PLT transfusion products. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem/progenitor cells derived from umbilical cord blood (UCB) units were used in a simple and efficient culture system to generate a cell product consisting of megakaryocytes (MKs) at different stages of development. The cultures thus generated were evaluated ex vivo and in vivo before and after cryopreservation. RESULTS: We generated a megakaryocytic cell product that can be cryopreserved without altering its phenotypical and functional capabilities. The infusion of such a product, either fresh or cryopreserved, into immune-deficient mice led to production of functional human PLTs which were observed within a week after infusion and persisted for 8 weeks, orders of magnitude longer than that observed after the infusion of traditional PLT transfusion products. The sustained human PLT engraftment was accompanied by a robust presence of human cells in the bone marrow (BM), spleen, and lungs of recipient mice. CONCLUSION: This is a proof-of-principle study demonstrating the creation of a cryopreservable megakaryocytic cell product which releases functional PLTs in vivo. Clinical development of such a product is currently being pursued for the treatment of thrombocytopenia in patients with hematological malignancies.

Effects of biomechanical forces on signaling in the cortical collecting duct (CCD).

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm(2) of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

Early response of alpha2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF-beta independent.

Acetaldehyde is fibrogenic and induces the expression of type I collagen genes in hepatic stellate cells. Some of these acetaldehyde-dependent events are mediated by H(2)O(2) and thus establish a direct connection between oxidative stress and collagen upregulation. We localized to the -378 to -183 region of the alpha2(I) collagen (COL1A2) promoter an acetaldehyde-responsive element (AcRE) functional in human hepatic stellate cells (HHSCs) and investigated molecular mechanisms whereby acetaldehyde stimulates and modulates its transcriptional activity. Because the AcRE co-localized with a previously described transforming growth factor beta (TGF-beta)1-responsive element, and both acetaldehyde and this cytokine induce their effects through H(2)O(2), we investigated whether all fibrogenic actions of acetaldehyde were mediated by this cytokine. Here we show that acetaldehyde-induced COL1A2 upregulation in HHSCs recognizes two distinct but overlapping early and late stages that last from 1 to 6 hours and from 6 to 24 hours, respectively. We present several lines of evidence to show that early acetaldehyde-mediated events are independent of TGF-beta1. These include significant time-course differences in the expression of COL1A2 and TGF-beta1 mRNAs and inability of neutralizing antibodies to TGF-beta1 to inhibit acetaldehyde-dependent collagen gene transcription and Smad 3 phosphorylation. We also show that although acetaldehyde-dependent upregulation of collagen was PI3K dependent, that of TGF-beta1 was PI3K independent. In conclusion, acetaldehyde-dependent mechanisms involved in COL1A2 upregulation are similar, but not identical, to those of TGF-beta1. We suggest that early acetaldehyde-dependent events induce the late expression of TGF-beta1 and create an H(2)O(2)-dependent autocrine loop that may sustain and amplify the fibrogenic response of this alcohol metabolite.

Identification of MoKA, a novel F-box protein that modulates Krüppel-like transcription factor 7 activity.

KLF7, a member of the Krüppel-like transcription factor family, is believed to regulate neurogenesis and cell cycle progression. Here, a yeast two-hybrid screen for KLF7 cofactors in the developing nervous system identified a novel 140-kDa protein named MoKA, for modulator of KLF7 activity. Interaction between MoKA and KLF7 was confirmed by the in vitro glutathione S-transferase pull-down assay and by coimmunoprecipitation of the proteins overexpressed in mammalian cells. Functional assays documented that MoKA is a KLF7 coactivator, and in situ hybridizations identified the developing nervous system and the adult testes as two sites of MoKA and Klf7 coexpression. Chromatin immunoprecipitation experiments demonstrated KLF7 binding to the p21(WAF1/Cip1) gene while transient transfection assays documented KLF7 stimulation of the p21(WAF1/Cip1) proximal promoter. Additional tests revealed that distinct structural motifs of MoKA direct interaction with KLF7 and shuttling between the nucleus and cytoplasm of asynchronously cycling cells. Altogether, our results strongly suggest that MoKA and KLF7 interact functionally to regulate gene expression during cell differentiation and identify the cell cycle regulator p21(WAF1/Cip1) as one of the targeted genes.