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BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.

DNA methylation, histone modifications, and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer. The development of drugs that target these processes is therefore important for cancer therapy. Inhibitors of DNA methylation and histone deacetylation have been approved by the Food and Drug Administration for treatment of hematologic malignancies. However, drugs that target other mechanisms still need to be developed. Recently, 3-deazaneplanocin A (DZNep) was reported to selectively inhibit trimethylation of lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) as well as reactivate silenced genes in cancer cells. This finding opens the door to the pharmacologic inhibition of histone methylation. We therefore wanted to further study the mechanism of action of DZNep in cancer cells. Western blot analysis shows that DZNep globally inhibits histone methylation and is not selective. Two other drugs, sinefungin and adenosine dialdehyde, have similar effects as DZNep on H3K27me3. Intriguingly, chromatin immunoprecipitation of various histone modifications and microarray analysis show that DZNep acts through a different pathway than 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. These observations give us interesting insight into how chromatin structure affects gene expression. We also determined the kinetics of gene activation to understand if the induced changes were somatically heritable. We found that upon removal of DZNep, gene expression is reduced to its original state. This suggests that there is a homeostatic mechanism that returns the histone modifications to their ground state" after DZNep treatment. Our data show the strong need for further development of histone methylation inhibitors."

Dendritic cell (DC) differentiation is regulated by stroma via a network of soluble and cell-bound factors. Notch is one of the major elements of this network. Its role in DC differentiation, however, is controversial. Here, we demonstrate that activation of Notch signaling in hematopoietic progenitor cells (HPCs) promoted differentiation of conventional DCs via activation of the canonical Wingless (Wnt) pathway. Inhibition of the Wnt pathway abrogated the effect of Notch on DC differentiation. The fact that activation of the Wnt pathway in Notch-1-deficient embryonic stem cells restored DC differentiation indicates that Wnt signaling is downstream of the Notch pathway in regulating DC differentiation. Notch signaling activated the Wnt pathway in HPCs via expression of multiple members of the Frizzled family of Wnt receptors, which was directly regulated by the CSL (RPB-Jkappa) transcription factor. Thus, these data suggest a model of DC differentiation via cooperation between Wnt and Notch pathways.

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways. The Wnt-beta-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation, but its role in the generation and maintenance of memory T cells is unknown. We found that induction of Wnt-beta-catenin signaling by inhibitors of glycogen sythase kinase-3beta or the Wnt protein family member Wnt3a arrested CD8(+) T cell development into effector cells. By blocking T cell differentiation, Wnt signaling promoted the generation of CD44(low)CD62L(high)Sca-1(high)CD122(high)Bcl-2(high) self-renewing multipotent CD8(+) memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of 'stemness' in mature memory CD8(+) T cells and have major implications for the design of new vaccination strategies and adoptive immunotherapies.

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here, we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore, Dax-1 knockdown induced upregulation of multilineage differentiation markers, and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis, we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly, the great majority of these genes are upregulated, showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells, as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis, respectively.

Progenitor cells have been extensively studied and therapeutically applied in tissue reconstructive therapy. Stromal vascular fraction (SVF) cells, which are derived from adipose tissue, may represent a potential source of the cells which undergo phenotypical differentiation into many lineages both in vitro as well as in vivo. The goal of this study was to check whether human SVF cells may differentiate into cardiomyocyte-like entities. Human SVF cells were induced to differentiate by their incubation in Methocult medium in the presence of SCF, IL-3 and IL-6. Morphological transformation of the cells was monitored using optical light microscope, whereas changes in expression of the genes typical for cardiac phenotype were measured by qRT-PCR. Incubation of the human SVF cells in the medium that promotes cardiomyocyte differentiation in vitro resulted in formation of myotubule-like structures accompanied by up-regulation of the myocardium-characteristic genes, such as GATA, MEF2C, MYOD1, but not ANP. Human SVF cells differentiate into cardiomyocyte-like cells in the presence of the certain set of myogenesis promoting cytokines.

Cytoplasmic APOBEC3G has been reported to block wild-type human immunodeficiency virus type 1 (HIV-1) infection in some primary cells. It is not known whether cytoplasmic APOBEC3G has residual activity in activated T cells, even though virion-packaged APOBEC3G does restrict HIV-1 in activated T cells. Because we found that APOBEC3G expression is greater in activated CD4(+) T-helper type 1 (Th1) lymphocytes than in T-helper type 2 (Th2) lymphocytes, we hypothesized that residual target cell restriction of incoming Vif-positive virions that lack APOBEC3G, if present, would be greater in Th1 than Th2 lymphocytes. Infection of activated Th1 cells with APOBEC3-negative virions did result in decreased amounts of early and late reverse transcription products and integrated virus relative to infection of activated Th2 cells. Two-long terminal repeat (2-LTR) circles, which are formed in the nucleus when reverse transcripts do not integrate, were increased after APOBEC3-negative virus infection of activated Th1 cells relative to infection of activated Th2 cells. In contrast, 2-LTR circle forms were decreased after infection of APOBEC3G-negative cells with APOBEC3G-containing virions relative to APOBEC3G-negative virions and with Th1 cell-produced virions relative to Th2 cell-produced virions. Increasing APOBEC3G in Th2 cells and decreasing APOBEC3G in Th1 cells modulated the target cell phenotypes, indicating causation by APOBEC3G. The comparison between activated Th1 and Th2 cells indicates that cytoplasmic APOBEC3G in activated Th1 cells partially restricts reverse transcription and integration of incoming Vif-positive, APOBEC3G-negative HIV-1. The differing effects of cytoplasmic and virion-packaged APOBEC3G on 2-LTR circle formation indicate a difference in their antiviral mechanisms.

The transcription factor T-bet (Tbx21) is critical for Th1 polarization of CD4(+) T cells. Genetic deletion of Tbx21 can cause either exacerbation or attenuation of different autoimmune diseases in animal models. In the nonobese diabetic (NOD) mouse, genetic deletion of the Ifng or the Il12b (IL-12p40) genes, which are both critical Th1 cytokines, does not reduce the incidence of autoimmune diabetes. These results suggest that autoimmune diabetes in the NOD may not be a Th1-driven disease. However, we report that Tbx21 deficiency in the NOD mouse completely blocks insulitis and diabetes due to defects both in the initiation of the anti-islet immune response and in the function of CD4(+) effector T cells. We find defective priming of naive islet-reactive T cells by the innate immune system in Tbx21(-/-) animals. By contrast to naive cells, activated islet-reactive BDC2.5 TCR-transgenic T cells do not require Tbx21 in recipient animals for efficient adoptive transfer of diabetes. However, when these BDC2.5 TCR-transgenic effector cells lack Tbx21, they are less effective at entering the pancreas and promoting diabetes than Tbx21(+/+) cells. Tbx21(-/-) regulatory T cells function normally in vitro and diabetes can be restored in Tbx21(-/-) mice by reducing regulatory T cell numbers. Thus, the absence of diabetes in the NOD.Tbx21(-/-) is due to intrinsic defects in both T cells and cells of the innate immune system paired with the relative preservation of regulatory T cell function.

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me(2)SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2x10(6) cells/mL. It was further established that DPSC can be stored at -85 degrees C or -196 degrees C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from textgreater85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.

Implantation of tissue engineering constructs is a promising technique to reconstruct injured tissue. However, after implantation the nutrition of the constructs is predominantly restricted to vascularization. Since cells possess distinct angiogenic potency, we herein assessed whether scaffold vitalization with different cell types improves scaffold vascularization. 32 male balb/c mice received a dorsal skinfold chamber. Angiogenesis, microhemodynamics, leukocyte-endothelial cell interaction and microvascular permeability induced in the host tissue after implantation of either collagen coated poly (L-lactide-co-glycolide) (PLGA) scaffolds (group 4), additionally seeded with osteoblast-like cells (OLCs, group 1), bone marrow mesenchymal stem cells (bmMSCs, group 2) or a combination of OLCs and bmMSCs (group 3) were analyzed repetitively over 14 days using intravital fluorescence microscopy. Apart from a weak inflammatory response in all groups, vascularization was found distinctly accelerated in vitalized scaffolds, indicated by a significantly increased microvascular density (day 6, group 1: 202+/-15 cm/cm(2), group 2: 202+/-12 cm/cm(2), group 3: 194+/-8 cm/cm(2)), when compared with controls (group 4: 72+/-5 cm/cm(2)). This acceleration was independent from the seeded cell type. Immunohistochemistry revealed in vivo VEGF expression in close vicinity to the seeded OLCs and bmMSCs. Therefore, the observed lack of cell type confined differences in the vascularization process suggests that the accelerated vascularization of vitalized scaffolds is VEGF-related rather than dependent on the potential of bmMSCs to differentiate into specific vascular cells.

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-beta (Abeta) protein, is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease, Down syndrome, and head injury. We recently reported that this protein, normally associated with neurodegenerative conditions, is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AbetaPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-beta, which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AbetaPP cleavage by beta-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression, an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AbetaPPalpha, which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AbetaPP is normally required for embryonic neurogenesis.

RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion, it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study, we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice, long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and beta(1) integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development, with mutant progenitors producing increased, abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc, the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus, Rbm15 appears to be required for normal HSC-niche interactions, for the ability of HSCs to contribute normally to adult hematopoiesis, and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc.