Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells.

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Inhibition of Digestive Enzymes and Antioxidant Activity of Extracts from Fruits of Cornus alba, Cornus sanguinea subsp. hungarica and Cornus florida-A Comparative Study.

The fruits of some Cornus species (dogwoods) are used in traditional medicine and considered potential anti-diabetic and hypolipemic agents. The aim of the study was to determine the ability of extracts from Cornus alba (CA), Cornus florida (CF), and Cornus sanguinea (CS) to inhibit digestive enzymes namely α-amylase, pancreatic lipase, and α-glucosidase, as well as isolation of compounds from plant material with the strongest effect. In addition, the phytochemical profile and antioxidant activity of extracts from three dogwoods were compared with HPLC-DAD-MS/MS and DPPH scavenging assay, respectively. Among the aqueous-ethanolic extracts, the activity of α-amylase was the most strongly inhibited by the fruit extract of CA (IC50 = 115.20 ± 14.31 µg/mL) and the activity of α-glucosidase by the fruit of CF (IC50 = 38.87 ± 2.65 µg/mL). Some constituents of CA fruit extract, such as coumaroylquinic acid, kaempferol, and hydroxytyrosol derivatives, were isolated. Among the three species of dogwood studied, the greatest biological potential was demonstrated by CA extracts, which are sources of phenolic acids and flavonoid compounds. In contrast, iridoid compounds or flavonoid glycosides found in fruits of CF or CS extracts do not play a significant role in inhibiting digestive enzymes but exert antioxidant activity.

Liposome-coated lipoplex-based carrier for antisense oligonucleotides.

The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL­2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

MicroRNA-125 family members exert a similar role in the regulation of murine hematopoiesis.

MicroRNAs (miRNAs) are crucial for proper functioning of hematopoietic stem and progenitor cells (HSPCs). Members of the miRNA-125 family (consisting of miR-125a, miR-125b1, and miR-125b2) are known to confer a proliferative advantage on cells upon overexpression, to decrease the rate of apoptosis by targeting proapoptotic genes, and to promote differentiation toward the myeloid lineage in mice. However, many distinct biological effects of the three miR-125 species have been reported as well. In the current study, we set out to assess whether the three miRNA-125s that carry identical seed sequences could be functionally different. Our data show that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. All miR-125 family members decreased the pool of phenotypically defined Lin(-)Sca(+)Kit(+)CD48(-)CD150(+) long-term hematopoietic stem cells, simultaneously increasing the self-renewal activity upon secondary transplantation. The downregulation of miR-125s in hematopoietic stem cells abolishes these effects and impairs long-term contribution to blood cell production. The introduction of a point mutation within the miRNA-125 seed sequence abolishes all abovementioned effects and leads to the restoration of normal hematopoiesis. Our results show that all miR-125 family members are similar in function, they likely operate in a seed-sequence-dependent manner, and they induce a highly comparable hematopoietic phenotype.

Sca-1 is an early-response target of histone deacetylase inhibitors and marks hematopoietic cells with enhanced function.

Histone deacetylase inhibitors (HDIs) have been shown to enhance hematopoietic stem and progenitor cell activity and improve stem cell outcomes after ex vivo culture. Identification of gene targets of HDIs is required to understand the full potential of these compounds and can allow for improved stem cell culturing protocols. The molecular process that underlies the biological effects of valproic acid (VPA), a widely used HDI, on hematopoietic stem/progenitor cells was investigated by studying the early-response genes of VPA. These genes were linked to VPA-induced enhancement of cell function as measured by in vitro assays. Genome-wide gene expression studies revealed over-representation of genes involved in glutathione metabolism, receptor and signal transducer activity, and changes in the hematopoietic stem/progenitor cells surface profile after short, 24-hour VPA treatment. Sca-1, a well-known and widely used stem cell surface marker, was identified as a prominent VPA target. We showed that multiple HDIs induce Sca-1 expression on hematopoietic cells. VPA strongly preserved Sca-1 expression on Lin(-)Sca1(+)ckit(+) cells, but also reactivated Sca-1 on committed progenitor cells that were Sca-1(neg), thereby reverting them to the Lin(-)Sca1(+)ckit(+) phenotype. We demonstrated that reacquired Sca-1 expression coincided with induced self-renewal capacity as measured by in vitro replating assays, while Sca-1 itself was not required for the biological effects of VPA as demonstrated using Sca-1-deficient progenitor cells. In conclusion, our results show that VPA modulates several genes involved in multiple signal transduction pathways, of which Sca-1 was shown to mark cells with increased self-renewal capacity in response to HDIs.

Hematopoietic stem cell expansion: challenges and opportunities.

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.

The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation.

Despite increasing knowledge on the regulation of hematopoietic stem/progenitor cell (HSPC) self-renewal and differentiation, in vitro control of stem cell fate decisions has been difficult. The ability to inhibit HSPC commitment in culture may be of benefit to cell therapy protocols. Small molecules can serve as tools to manipulate cell fate decisions. Here, we tested 2 small molecules, valproic acid (VPA) and lithium (Li), to inhibit differentiation. HSPCs exposed to VPA and Li during differentiation-inducing culture preserved an immature cell phenotype, provided radioprotection to lethally irradiated recipients, and enhanced in vivo repopulating potential. Anti-differentiation effects of VPA and Li were observed also at the level of committed progenitors, where VPA re-activated replating activity of common myeloid progenitor and granulocyte macrophage progenitor cells. Furthermore, VPA and Li synergistically preserved expression of stem cell-related genes and repressed genes involved in differentiation. Target genes were collectively co-regulated during normal hematopoietic differentiation. In addition, transcription factor networks were identified as possible primary regulators. Our results show that the combination of VPA and Li potently delays differentiation at the biologic and molecular levels and provide evidence to suggest that combinatorial screening of chemical compounds may uncover possible additive/synergistic effects to modulate stem cell fate decisions.

Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells.

Hematopoietic stem/progenitor cell (HSPC) traits differ between genetically distinct mouse strains. For example, DBA/2 mice have a higher HSPC frequency compared with C57BL/6 mice. We performed a genetic screen for micro-RNAs that are differentially expressed between LSK, LS(-)K(+), erythroid and myeloid cells isolated from C57BL/6 and DBA/2 mice. This analysis identified 131 micro-RNAs that were differentially expressed between cell types and 15 that were differentially expressed between mouse strains. Of special interest was an evolutionary conserved miR cluster located on chromosome 17 consisting of miR-99b, let-7e, and miR-125a. All cluster members were most highly expressed in LSKs and down-regulated upon differentiation. In addition, these microRNAs were higher expressed in DBA/2 cells compared with C57BL/6 cells, and thus correlated with HSPC frequency. To functionally characterize these microRNAs, we overexpressed the entire miR-cluster 99b/let-7e/125a and miR-125a alone in BM cells from C57BL/6 mice. Overexpression of the miR-cluster or miR-125a dramatically increased day-35 CAFC activity and caused severe hematopoietic phenotypes upon transplantation. We showed that a single member of the miR-cluster, namely miR-125a, is responsible for the majority of the observed miR-cluster overexpression effects. Finally, we performed genome-wide gene expression arrays and identified candidate target genes through which miR-125a may modulate HSPC fate.