Carmen Ka Yee Chuen

Promoting effects of serotonin on hematopoiesis : Ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5‐hydroxytryptamine (5‐HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony‐forming unit‐fibroblast (CFU‐F) formation, and antiapoptosis of megakaryoblastic M‐07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony‐forming unit‐mixed [CFU‐GEMM]) and multilineage committed progenitors (burst‐forming unit/colony‐forming unit‐erythroid [BFU/CFU‐E], colony‐forming unit‐granulocyte macrophage, colony‐forming unit‐megakaryocyte, CD61+CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU‐E, and CFU‐GEMM engraftment in BM of animals 6 weeks post‐transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet‐derived growth factor, or vascular endothelial growth factor stimulated BM CFU‐F formation. In M‐07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase‐3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5‐HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation.

Small peptide analogue of SDF-1α supports survival of cord blood CD34+ cells in synergy with other cytokines and enhances their ex vivo expansion and engraftment into nonobese diabetic/severe combined immunodeficient mice

The SDF‐1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF‐1 peptide analogue CTCE‐0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE‐0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt‐3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE‐0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38− cells, colony‐forming unit‐mixed [CFU‐GEMMs]), erythroid (CFU‐Es), myeloid (CFU‐GMs), and megakaryocytic (CD61+CD41+ cells, CFU‐MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE‐0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF‐1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.

Platelet-derived growth factor up-regulates the expression of transcription factors NF-E2, GATA-1 and c-Fos in megakaryocytic cell lines

Platelet-derived growth factor (PDGF) is a platelet alpha-granule protein. In previous reports, we demonstrated the expression of PDGF receptors on platelets and megakaryocytic cells and that PDGF enhanced the proliferation of megakaryocytic progenitor cells. In this study, we investigated the effects of PDGF on mRNA and protein expressions of megakaryocyte-associated transcription factors, c-Fos, GATA-1, NF-E2 and PU.1, in two human megakaryocytic cell lines CHRF-288-11 and DAMI. RT-PCR/Southern blot analysis and Real-time PCR demonstrated that PDGF increased the mRNA expression of c-Fos, GATA-1 and NF-E2, but not PU.1 in a dose- and time-dependent manner. The activation was confirmed at the protein level by Western blot analysis of both total cell and nuclear lysates. The addition of increasing concentrations of Tyrphostin AG1295, an inhibitor of PDGF receptor kinase, blocked the stimulatory effect of PDGF on the mRNA and protein expressions of these transcription factors. The up-regulation of c-Fos, GATA-1 and NF-E2 protein by PDGF was inhibited by actinomycin D and cycloheximide, suggesting that mRNA and protein synthesis might be involved in the mechanism. Our data suggest a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions and we speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis.

Preclinical ex vivo expansion of G‐CSF‐mobilized peripheral blood stem cells: effects of serum‐free media, cytokine combinations and chemotherapy

Abstract:  Objectives: Ex vivo expansion of granulocyte‐colony stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSC) is a promising approach for overcoming the developmental delay of bone marrow (BM) reconstitution after transplantation. This project investigated the effects of culture duration, serum‐free media, cytokine combinations, and chemotherapy on the outcomes of expansion. Methods: Enriched CD34+ cells were cultured for 8 or 10 d in serum‐free media (QBSF‐60 or X‐Vivo 10) and four combinations of cytokines consisting of recombinant human pegylated‐megakaryocyte growth and development factor, stem cell factor, flt‐3 ligand, G‐CSF, interleukin (IL)‐6, platelet‐derived growth factor (PDGF), and IL‐1β. Results: Eight days of culture in QBSF‐60 significantly supported efficient expansions of CD34+ cells, CD34+ CD38− cells, colony‐forming units (CFU) of myeloid, erythroid, megakaryocytic, and mixed lineages to 3.76‐, 14.4‐, 28.3‐, 24.0‐, 38.1‐, and 15.7‐fold, respectively. Whilst PDGF or IL‐6 enhanced the expansion of early, myeloid, and erythroid progenitors, IL‐1β specifically promoted the megakaryocytic lineage. Engraftment of human CD45+ cells were detectable in all non‐obese diabetic/severe‐combined immunodeficient mice transplanted with expanded PBSC from donor samples, being 5.80 ± 3.34% of mouse BM cells. The expansion and engraftment capacity of CD34+ cells from subjects postchemotherapy were significantly compromised across the panel of progenitor cells. Conclusion: Our results provided an optimized protocol for PBSC expansion, applicable to ameliorating neutropenia and thrombocytopenia in post‐BM transplant patients by the prompt provision of progenitor cells. For postchemotherapy patients, expansion products might provide committed progenitors for improving short‐term engraftment, but not self‐renewable stem cells.

The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances their ex vivo expansion

Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self‐renewable stem cells. This study investigated the effect of a mannose‐binding lectin, NTL, purified from Narcissus tazetta var. chinensis, on prolonged maintenance and expansion of cord blood CD34+ cells. Our results showed that the presence of NTL or Flt‐3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum‐ and cytokine‐free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34+ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL‐enhanced expansion of early progenitors (CD34+, CD34+CD38−, mixed colony‐forming units and CFU‐GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst‐forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d‐expanded cells to non‐obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45+ cells was observed in the bone marrow of animals that received NTL‐treated cells. The dual functions of NTL on long‐term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.

Expression of interleukin (IL) 1 type I and type II receptors in megakaryocytic cells and enhancing effects of IL-1β on megakaryocytopoiesis and NF-E2 expression: IL-1β and Megakaryocytopoiesis

Megakaryocytopoiesis is regulated by thrombopoietin (TPO) and cytokines such as interleukin 3 (IL‐3), IL‐6 and IL‐11. This study investigated the in vitro effects of IL‐1β on megakaryocytopoiesis and the expression of IL‐1 type I and type II receptors (IL‐1 RI and RII) on megakaryocytic cell lines and primary cells. Our results demonstrated that IL‐1β alone or in combination with TPO induced megakaryocyte colony forming units (CFU‐MK) from murine and human haematopoietic cells. Using reverse‐transcription polymerase chain reaction (RT‐PCR) and Southern hybridization techniques, the mRNA of IL‐1β, IL‐1 RI, IL‐1 RII and the transcription factor NF‐E2 were detected in CD61+CD41+ cells cultured from cord blood and four megakaryocytic cell lines, Meg‐01, DAMI, CHRF‐288–11 and M‐07e. The expression of IL‐1 RI and RII proteins was confirmed by flow cytometry and immunofluorescence staining. In Meg‐01 cells, the expression of NF‐E2 was increased at both mRNA and protein levels after treatment with IL‐1β for 4 h. This study demonstrated for the first time the presence of IL‐1 receptors on megakaryocytic cells and the induction of NF‐E2 by IL‐1β. The mitogenic effect of IL‐1β on this lineage could be mediated through IL‐1 receptors and the activation of NF‐E2.