Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

[Truncated abstract] Human complement receptor 2 (CR2/CD21) is a cell surface glycoprotein detected on specific cells involved in immunity, which binds complement C3 cleavage fragments, cellular ligands IFN-? and CD23 as well as the EBV coat protein, gp350/220. During the early stages of B-cell development CR2/CD21 is silenced. Expression is initiated on immature B-cells escaping negative selection. During peripheral maturation CR2/CD21 is up-regulated with B-cell sub-populations showing distinctive surface levels (comparatively low, intermediate or high). CR2/CD21 is silenced upon terminal plasmacytic differentiation. Appropriate timing and expression level of CR2/CD21 is important for the development of a healthy B-cell repertoire. Previous studies have identified sequences within the proximal promoter and first intron of CR2/CD21 that cooperate within native chromatin to control cell-specific silencing. Further, analysis of cultured human cells has revealed chromatin structural variation causing DNase I hypersensitivity at these regulatory sites in a CR2/CD21-expressing mature B-cell line (Raji) which are absent in a non-lymphoid cell type (K562). The primary focus of the present study involved characterising chromatin structural variation over previously recognized DNase I hypersensitive regions at the CR2/CD21 locus in human cells to understand how chromatin structure might regulate developmental expression of CR2/CD21. ... These studies provide evidence that notch signaling influences CR2/CD21 expression in human cell lines. First, in vivo binding of CBF1 to CR2/CD21 sequences in the proximal promoter and CRS implies that CR2/CD21 is a direct target of notch activation. Second, the effect of exogenous notch signalling molecules on CR2/CD21 proximal promoter activity was modulated by factors binding tandem E-boxes near the transcriptional start site suggesting that the notch pathway may also influence CR2/CD21 expression via control of HLH molecules. Third, initiation of CR2/CD21 expression was observed in a nonexpressing pre-B cell line (Reh) by co-culture with stromal cells expressing a notch ligand (OP9-DL) but not control stroma (OP9-GFP). Together, these findings support a role for notch regulation of B-cell maturation and invite speculation that initiation of CR2/CD21 expression following negative selection of immature B-cells involves crosstalk between HLH transcriptional regulators and the notch pathway. Furthermore, the Reh/OP9-DL co-culture system may provide a model to directly study the relationship between cell signalling molecules, transcription factor regulation, chromatin structural variation and differentiation of B-cells.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2009. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 29, 2009). Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.

Orientador: Teresa Cristina Cardoso da Silva / Banca: Roberto Gameiro de Carvalho / Banca: Andréa Fontes Garcia / Resumo: As células mesenquimais derivadas da geleia de Wharton isoladas da matriz do cordão umbilical canino tem sido sugeridas como uma fonte promissora de MSCs para serem usadas nas aplicações clínicas em ciência veterinária, como uma ferramenta potencialmente efetiva na regeneração óssea. MicroRNA (miARN) é um regulador pós-transcricional da expressão gênica em várias condições fisiológicas, incluindo a osteogênese. Neste estudo, as MSCs caninos (cMSCs) isoladas da geléia de Wharton foram induzidos a osteogênese e a transcrição de miR-106b foi avaliada em 0, 7, 14 e 21 dias após a indução. Em outro experimento, as cMSC foram transfectadas com um imitador de miR106b e um inibidor e induzidos a osteogênese. Morfologicamente, cMSCs transfectadas com um inibidor de miR-106b produziram células semelhantes a osteócitos quando comparadas às mesmas células transfectadas com o mímico de miR-106b. cMSCs apresentaram transcrição de miR-106b após 7 dias de osteoindução em um nível baixo em comparação com o controle positivo, enquanto as células transfectadas com o mímico de miR-106b mostraram que o miR-106b deveria ser regulado positivamente. Após a inibição da expressão de miR-106b em cMSCs osteoinduzidas, a atividade da fosfatase alcalina (ALP) foi aumentada. A transcrição do mRNA de osteocalcina, osteopontina e RUNX2 foi regulada positivamente aos 21 dias após a osteoindução, após a inibição de miR106b. Esses achados, pela primeira vez, mostraram que a expressão de miR106b regula negativam... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Wharton's jelly derived-MSCs isolated from canine umbilical cord matrix have been suggested as a promising source of MSCs to be used for clinical applications in veterinary science, as a potentially effective tool in bone regeneration. MicroRNA (miRNA) is a post-transcriptional regulator of gene expression in several physiological conditions, including osteogenesis In this study, canine MSCs (cMSCs) isolated from Wharton's jelly were induced to osteogenesis and miR-106b transcription was measured at 0, 7, 14 and 21 days following induction. In another experiment, cMSCs were transfected with a miR106b mimic and an inhibitor and induced to osteogenesis. Morphologically, cMSCs transfected with an inhibitor of miR-106b appeared as osteocyte-like cells when compared to the same cells transfected with the mimic of miR-106b. cMSCs showed miR-106b transcription after 7 days of osteoinduction was at a low level compared to the positive control, whereas transfected cells with the miR-106b mimic showed miR-106b to be upregulated. After inhibition of miR-106b expression in osteoinduced cMSCs, alkaline phosphatase (ALP) activity was increased. Osteocalcin, osteopontin and RUNX2 mRNA transcription were upregulated at 21 days after osteoinduction following miR-106b inhibition. These findings have, for the first time, shown that the expression of miR-106b negatively regulates osteogenesis in canine MSCs derived from Wharton's jelly and seems to interfere with cell differentiation. / Mestre