Extent Of Cell Expansion Research Articles

A small-molecule/cytokine combination enhances hematopoietic stem cell proliferation via inhibition of cell differentiation

BackgroundAccumulated evidence supports the potent stimulating effects of multiple small molecules on the expansion of hematopoietic stem cells (HSCs) which are important for the therapy of various hematological disorders. Here, we report a novel, optimized formula, named the SC cocktail, which contains a combination of three such small molecules and four cytokines.MethodsSmall-molecule candidates were individually screened and then combined at their optimal concentration with the presence of cytokines to achieve maximum capacity for stimulating the human CD34+ cell expansion ex vivo. The extent of cell expansion and the immunophenotype of expanded cells were assessed through flow cytometry. The functional preservation of HSC stemness was confirmed by additional cell and molecular assays in vitro. Subsequently, the expanded cells were transplanted into sublethally irradiated NOD/SCID mice for the assessment of human cell viability and engraftment potential in vivo. Furthermore, the expression of several genes in the cell proliferation and differentiation pathways was analyzed through quantitative polymerase chain reaction (qPCR) during the process of CD34+ cell expansion.ResultsThe SC cocktail supported the retention of the immunophenotype of hematopoietic stem/progenitor cells remarkably well, by yielding purities of 86.6 ± 11.2% for CD34+ cells and 76.2 ± 10.5% for CD34+CD38– cells, respectively, for a 7-day culture. On day 7, the enhancement of expansion of CD34+ cells and CD34+CD38– cells reached a maxima of 28.0 ± 5.5-fold and 27.9 ± 4.3-fold, respectively. The SC cocktail-expanded CD34+ cells preserved the characteristics of HSCs by effectively inhibiting their differentiation in vitro and retained the multilineage differentiation potential in primary and secondary in vivo murine xenotransplantation trials. Further gene expression analysis suggested that the small-molecule combination strengthened the ability of the cytokines to enhance the Notch pathway for the preservation of HSC stemness, and inhibited the ability of the cytokines to activate the Wnt pathway for HSC differentiation.ConclusionsWe developed an optimal small-molecule/cytokine combination for the enhancement of HSC expansion via inhibition of differentiation. This approach indicates promising application for preparation of both the HSCs and the mature, functional hematopoietic cells for clinical transplantation.

Small Molecule Combination Enhances CD34+/CD38- Cell Proliferation Via Inhibition of Differentiation

Hematopoietic stem cells (HSCs) have become increasingly attractive for the therapy of various hematological system disorders. The aim of this study is to identify approaches that promote the expansion of HSCs. We present here the identification of a combination of small molecules and cytokines that is effective in retaining high stemness of hematopoietic stem/progenitor cells while promoting cell proliferation by inhibiting differentiation.Firstly, five small-molecule candidates were screened for their individual effects on ex vivo expansion of human peripheral blood CD34+ cells in the presence of selected cytokines. The best compounds at their optimal concentrations were further analyzed in combination, to achieve maximum capacity for stimulating the CD34+CD38- cell expansion ex vivo. The extent of cell expansion and the immunophenotype of expanded cells were assessed through flow cytometry. Additional cell and molecular assays were performed to confirm that the expanded CD34± cells are functionally normal in vitro. Subsequently, the expanded cells were transplanted into sublethally irradiated NOD/SCID mice for the assessment ofhuman cell viability and engraftment potential in vivo. Furthermore, the expression of several genes in the cell proliferation and differentiation pathways was analyzed through qPCR during the process of CD34±cell expansion.Following multiple rounds of screening, an optimal formula (named as "SVC cocktail") was obtained, which consisted of four cytokines (stem cell factor, flt-3 ligand, thrombopoietin and interleukin-6) and three small molecules (Stem Regenin 1, valproic acid and CAY10433). CD34+ cells cultured with SVC cocktail had a purity of 76.2%±7.5% and reached expansion folds of 27.9±4.3 for CD34+/CD38- HSCs on day 7. In contrast, CD34+ cells cultured with the cytokines alone displayed a purity of 27.4%±6.3% and expansion folds of 15.5±2.2 for CD34+/CD38- cells. The groups with small molecules only (plus DMSO, the vehicle), or with basal medium only, showed no surviving cells on day 4. Furthermore, cell cycle analysis indicated that the SVC cocktail-induced CD34+/CD38- cells stayed in a more quiescent state (G0/G1: 75.2%±3.6%; S: 9.2%±2.4%). On the other hand, the cells cultured without the three small molecules had active DNA synthesis (G0/G1: 56.0%±2.0%; S: 31.8%±3.2%), implicating a trend of enhanced cell differentiation in the cytokine alone group. RT-qPCR analysis further demonstrated that the expression of HSC stemness markers CD90, CD133, CD117, ALDH1, Bmi1, HoxB4, GATA-2, Runx1, and CXCR4 were elevated in the SVC cocktail-induced CD34+ cells, but dramatically reduced or barely detectable in the cytokine alone group. In addition, CFU assays for the SVC cocktail group vs the cytokine alone group demonstrated BFU-E of 54.0±4.6 vs 11.7±1.5, CFU-GM of 71.0±2.7 vs 8.3±2.5, CFU-GEMM of 40.7±3.8 vs 5.0±2.0 and CFU-Mk of 6.7±1.5 vs 0.7±0.6, respectively. For the in vivo engraftment in mouse bone marrow, human CD45 rate in the SVC cocktail group was much higher than in the cytokine alone group (21.1%±2.7% vs 0.5%±0.1%); similar group differences were also found in the CD34+ and CD34+CD38- rate (7.7%±1.4% vs 1.6%±1.2% and 6.8%±2.2% vs 1.6%±0.1% respectively), all at 8 weeks post transplantation. Moreover, qPCR analysis of Notch and Wnt signaling pathways for cultured cells on day 7 showed that the expression of Notch target genes (related to high activation of HSC property) was enhanced in the SVC cocktail group compare to the cytokine group (HES5: 9.2±2.3 vs 3.6±1.4 in arbitrary units; HEY1: 6.3±1.9 vs 2.6±1.2; HES1: 3.2±1.3 vs 1.3±0.4; Notch1: 1.4±0.3 vs 1.2±0.3), whereas the expression of Wnt target genes (related to activation of HSC differentiation) was greater in the cytokine alone group than in the SVC cocktail group (CCND1: 10.1±4.3 vs 1.2±0.8; LEF1: 4.3±0.6 vs 2.9±0.2; PPAR D: 3.4±0.3 vs 1.5±0.1; FZD2: 1.8±0.2 vs 1.0±0.1).Taken together, our results show that the new SVC cocktail is able to retain the characteristics of HSCs remarkably well, by enhancing their expansion while inhibiting their differentiation. Mechanistically, it appears that the three small molecules can effectively inhibit the cytokines' pro-differentiation effects on CD34+CD38- cells without affecting the cytokines' ability to stimulate cell proliferation. DisclosuresWang:Biopharmagen Corp.: Employment. Ren:Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

Alterations in auxin homeostasis suppress defects in cell wall function.

The plant cell wall is a highly dynamic structure that changes in response to both environmental and developmental cues. It plays important roles throughout plant growth and development in determining the orientation and extent of cell expansion, providing structural support and acting as a barrier to pathogens. Despite the importance of the cell wall, the signaling pathways regulating its function are not well understood. Two partially redundant leucine-rich-repeat receptor-like kinases (LRR-RLKs), FEI1 and FEI2, regulate cell wall function in Arabidopsis thaliana roots; disruption of the FEIs results in short, swollen roots as a result of decreased cellulose synthesis. We screened for suppressors of this swollen root phenotype and identified two mutations in the putative mitochondrial pyruvate dehydrogenase E1α homolog, IAA-Alanine Resistant 4 (IAR4). Mutations in IAR4 were shown previously to disrupt auxin homeostasis and lead to reduced auxin function. We show that mutations in IAR4 suppress a subset of the fei1 fei2 phenotypes. Consistent with the hypothesis that the suppression of fei1 fei2 by iar4 is the result of reduced auxin function, disruption of the WEI8 and TAR2 genes, which decreases auxin biosynthesis, also suppresses fei1 fei2. In addition, iar4 suppresses the root swelling and accumulation of ectopic lignin phenotypes of other cell wall mutants, including procuste and cobra. Further, iar4 mutants display decreased sensitivity to the cellulose biosynthesis inhibitor isoxaben. These results establish a role for IAR4 in the regulation of cell wall function and provide evidence of crosstalk between the cell wall and auxin during cell expansion in the root.

Chronic Hepatitis C Virus Infection Breaks Tolerance and Drives Polyclonal Expansion of Autoreactive B Cells

Chronic Hepatitis C virus (HCV) infection has been linked with B cell lymphoproliferative disorders and several autoimmune-related diseases. The mechanisms of how chronic viral infection affects B cell development and predisposes the patients to autoimmune manifestations are poorly understood. In this study, we established an experimental system to probe the B cell responses and characterize the antibodies from chronic-HCV-infected individuals. We identified an unusual polyclonal expansion of the IgM memory B cell subset in some patients. This B cell subset is known to be tightly regulated, and autoreactive cells are eliminated by tolerance mechanisms. Genetic analysis of the immunoglobulin (Ig) heavy chain variable gene (V(H)) sequences of the expanded cell population showed that the levels of somatic hypermutation (SHM) correlate with the extent of cell expansion in the patients and that the V(H) genes exhibit signs of antigen-mediated selection. Functional analysis of the cloned B cell receptors demonstrated autoreactivity in some of the expanded IgM memory B cells in the patients which is not found in healthy donors. In summary, this study demonstrated that, in some patients, chronic HCV infection disrupts the tolerance mechanism that normally deletes autoreactive B cells, therefore increasing the risk of developing autoimmune antibodies. Long-term follow-up of this expanded B cell subset within the infected individuals will help determine whether these cells are predictors of more-serious clinical manifestations.

Funneling of gibberellin signaling by the GRAS transcription regulator SCARECROW-LIKE 3 in the Arabidopsis root

During plant development, because no cell movement takes place, control of the timing and extent of cell division and coordination of the direction and extent of cell expansion are particularly important for growth and development. The plant hormone gibberellins (GAs) play key roles in the control of these developmental processes. However, little is known about the molecular components that integrate the generic GA signaling into a specific cell/tissue to coordinate cell division and cell expansion. Here we report that scarecrow-like 3 (SCL3), a GRAS protein, acts as a positive regulator to integrate and maintain a functional GA pathway by attenuating the DELLA repressors in the root endodermis. The tissue-specific maintenance of GA signaling in the root endodermis plays distinct roles along the longitudinal root axis. While in the elongation/differentiation zone (EDZ), the endodermis-confined GA pathway by SCL3 controls primarily coordination of root cell elongation; in the meristem zone (MZ) SCL3 in conjunction with the short-root/scarecrow (SHR/SCR) pathway controls GA-modulated ground tissue maturation. Our findings highlight the regulatory network of the GRAS transcription regulators (SCL3, DELLAs, and SHR/SCR) in the root endodermis, shedding light on how GA homeostasis is achieved and how the maintenance of GA signaling controls developmental processes in roots.

The Role of Receptor-Like Kinases in Regulating Cell Wall Function

The plant cell wall is a rigid but highly dynamic structure that provides mechanical support, protection against pathogen attack, and determines the direction and extent of cell expansion ([Humphrey et al., 2007][1]). The dynamic nature of the plant cell wall allows growing cells to expand while

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.

A Small Gem with Great Powers: Geminin Keeps Neural Progenitors Thriving

Timing and extent of cell expansion and specialization in the developing nervous system are tightly controlled. In a recent issue of Genes and Development, Seo and coworkers (2005a) show that geminin (Gem), a protein involved in cell cycle control, also regulates the transition from proliferating neural progenitors to differentiating neurons.

Disruptions of the Arabidopsis Enoyl-CoA Reductase Gene Reveal an Essential Role for Very-Long-Chain Fatty Acid Synthesis in Cell Expansion during Plant Morphogenesis

In the absence of cell migration, plant architecture is largely determined by the direction and extent of cell expansion during development. In this report, we show that very-long-chain fatty acid (VLCFA) synthesis plays an essential role in cell expansion. The Arabidopsis thaliana eceriferum10 (cer10) mutants exhibit severe morphological abnormalities and reduced size of aerial organs. These mutants are disrupted in the At3g55360 gene, previously identified as a gene coding for enoyl-CoA reductase (ECR), an enzyme required for VLCFA synthesis. The absence of ECR activity results in a reduction of cuticular wax load and affects VLCFA composition of seed triacylglycerols and sphingolipids, demonstrating in planta that ECR is involved in all VLCFA elongation reactions in Arabidopsis. Epidermal and seed-specific silencing of ECR activity resulted in a reduction of cuticular wax load and the VLCFA content of seed triacylglycerols, respectively, with no effects on plant morphogenesis, suggesting that the developmental phenotypes arise from abnormal sphingolipid composition. Cellular analysis revealed aberrant endocytic membrane traffic and defective cell expansion underlying the morphological defects of cer10 mutants.

Interleukin-2 in neuroblastoma: clinical perspectives based on biological studies.

Stage IV neuroblastoma (NB) is a disease with a poor prognosis. Chemotherapeutical intensification and hematological rescue with autologous bone marrow transplantation (ABMT) achieve some complete remissions (CR), but most patients relapse during the first year. Immunotherapy could be an alternative in this situation of high risk of relapse due to residual disease and ABMT-related immunodepression. Ten stage IV NB patients in CR or very good partial remission have been treated with recurrent 5-day cycles of high doses of Interleukin-2 (IL2) after ABMT throughout one year (usually 5-6 cycles). Natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activities, as well as phenotype and number of circulating NK cells were determined, before and after each course of IL2 treatment. The effects promoted by IL2 varied during treatment: early cycles of IL2 induced a great extent of cell expansion, mainly on CD3-/CD16-/CD56+bright and CD8+dim cell phenotypes; conversely, late courses of IL2 promoted higher NK cytotoxic activity but a lesser increase on circulating NK cells. The induction of LAK activity did not significantly differ from early and late IL2 treatments. Clinical results are still inconclusive due to the small number of patients. The median follow-up of patients treated with IL2 is 24 months and the disease free survival (DFS) probability is 0.80 +/- 0.12 vs 0.16 +/- 0.15 from a historical control with identical treatment, but in the absence of IL2 treatment (p < 0.005). IL2 treatment-related toxicity was mild and no interruption of the treatment was required. Extremely accurate hydric control was carried out to avoid, as much as possible, the consequences of vascular leak syndrome, one of the most important toxic effects of IL2 treatment. The results presented here suggest an evolution of NK activity during IL2 treatment after ABMT, which should be taken into account for the designing of new immunotherapeutical protocols and opens a promising perspective in treatment of stage IV neuroblastoma.