Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45−) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool

Modeling SDF-1-induced mobilization in leukemia cell lines

The stromal cell-derived factor 1 (SDF-1) is essential for circulation, homing, and retention of hematopoietic stem cells in the bone marrow. Present evidence indicates that this factor might play an important role in leukemia cells as well. The aim of this study is to present a model of SDF-1-induced mobilization using leukemia cell lines. CXCR4 expression was compared in Kasumi-1, Jurkat, HL-60, KG-1a, and K562 cells by flow cytometry and Western blot. Migration was analyzed with Transwell assays, and adhesive cell-cell interaction was quantified with a standardized adhesion assay and flow cytometry. CXCR4 was expressed by all leukemic cell lines analyzed, although surface expression of this receptor was found in Kasumi-1 and Jurkat cells only. Correspondingly, SDF-1? effects on migration and cell-cell adhesion were observed in Kasumi-1 and Jurkat cells only, and this could be blocked by AMD3100 in a reversible manner. We have provided evidence that SDF-1? acts as a chemotactic and chemokinetic agent. In addition, surface expression of integrin-?2, activated leukocyte cell adhesion molecule and N-cadherin decreased after stimulation with SDF-1?. SDF-1? affects cell-cell adhesion and migration only in leukemia cells on which the CXCR4 receptor is present on the surface. An SDF-1 gradient is not necessarily required to induce migration, as chemokinesis can also occur. Upon stimulation with SDF-1, CXCR4 promotes modifications on the surface pattern of adhesion molecules, which have an influence on adhesion and migration. � 2012 ISEH - Society for Hematology and Stem Cells

Supplementary Material for: A Novel Homozygous <b><i>KLHL3</i></b> Mutation as a Cause of Autosomal Recessive Pseudohypoaldosteronism Type II Diagnosed Late in Life

Introduction: Pseudohypoaldosteronism type II (PHA II) is a Mendelian disorder, featuring hyperkalemic acidosis and low plasma renin levels, typically associated with hypertension. Mutations in WNK1, WNK4, CUL3, and KLHL3 cause PHA II, with dominant mutations in WNK1, WNK4, and CUL3 and either dominant or recessive mutations in KLHL3. Fourteen families with recessive KLHL3 mutations have been reported, with diagnosis at the age of 3 months to 56 years, typically in individuals with normal kidney function. Methods: We performed clinical and genetic investigations in a patient with hyperkalemic hypertension and used molecular dynamics simulations, heterologous expression in COS7 cells, and Western blotting to investigate the effect of a KLHL3 candidate disease mutation on WNK4 protein expression. Results: The patient, a 58-year-old woman from a consanguineous family, showed hypertension, persistent hyperkalemic acidosis associated with severe muscle pain, nephrolithiasis, chronic kidney disease (CKD), and coronary heart disease. Therapy with hydrochlorothiazide corrected hyperkalemia, hypertension, and muscle pain. Genetic analysis revealed a homozygous p.Arg431Trp mutation at a highly conserved KLHL3 position. Simulations suggested reduced stability of the mutant protein, which was confirmed by Western blot. Compared with wild-type KLHL3, cotransfection of p.Arg431Trp KLHL3 led to increased WNK4 protein levels, inferred to cause increased NaCl reabsorption via the thiazide-sensitive carrier and PHA II. Conclusions: Even in patients presenting late in life and in the presence of CKD, PHA II should be suspected if renin levels are low and hyperkalemic acidosis and hypertension are inadequate for CKD stage, particularly in the presence of a suspicious family history