The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development.

Unrestricted somatic stem cells (USSCs) have been recently identifi ed in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confi rmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (≥2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This fi nding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

BackgroundATP-binding cassette (ABC) transporters protect cells against unrelated (toxic) substances by pumping them across cell membranes. Earlier we showed that many ABC transporters are highly expressed in hematopoietic stem cells (HSCs) compared to more committed progenitor cells. The ABC transporter expression signature may guarantee lifelong protection of HSCs but may also preserve stem cell integrity by extrusion of agents that trigger their differentiation. Here we have studied whether non-hematopoietic stem cells (non-HSCs) exhibit a similar ABC transporter expression signature as HSCs.ResultsABC transporter expression profiles were determined in non-hematopoietic stem cells (non-HSCs) from embryonic, neonatal and adult origin as well as in various mature blood cell types. Over 11,000 individual ABC transporter expression values were generated by Taqman Low Density Arrays (TLDA) to obtain a sensitivity comparable with quantitative real-time polymerase chain reactions. We found that the vast majority of transporters are significantly higher expressed in HSCs compared to non-HSCs. Furthermore, regardless their origin, non-HSCs exhibited strikingly similar ABC transporter expression profiles that were distinct from those in HSCs. Yet, sets of transporters characteristic for different stem cell types could be identified, suggesting restricted functions in stem cell physiology. Remarkably, in HSCs we could not pinpoint any single transporter expressed at an evidently elevated level when compared to all the mature blood cell types studied.ConclusionsThese findings challenge the concept that individual ABC transporters are implicated in maintaining stem cell integrity. Instead, a distinct ABC transporter expression signature may be essential for stem cell function. The high expression of specific transporters in non-HSCs and mature blood cells suggests a specialized, cell type dependent function and warrants further functional experiments to determine their exact roles in cellular (patho)physiology.

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1B Q287* , cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 – REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1B Q287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1B Q287* -induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1B Q287* including cell division and interferon signaling. Proteome studies on platelets from GFI1B Q287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1B Q287* deregulates this program through LSD1-RCOR-HDAC sequestering.