Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

Cognitive functions and normal tension glaucoma

Purpose: Only a few studies have analyzed the potential link between glaucoma and cognitive function impairment. They have found controversial results. This study aims to perform quick cognitive function assessment with clock drawing test (CDT) using two different scoring systems and compare between normal tension glaucoma (NTG) and cataract patients. Methods: Totally, 30 NTG and 30 patients with cataracts were included in a prospective, pilot study. The predrawn circle was given, and patients were asked to draw the clock showing a time of 11:10. The test was evaluated using two methods – Freund method using a 7-point scoring scale (optimal cutoff ≤4) and Rakusa using a 4-point scoring scale (optimal cutoff ≤3). The level of significance was set at P < 0.05. Results: CDT result was significantly better in cataract group than in NTG group: 3.5 (2) versus 2 (2) by Freund, (P = 0.003) and 6.5 (1) versus 4.5 (2.75) by Rakusa, respectively (P = 0.004). Sixty percent (n = 18) of NTG group and 10% (n = 3) of cataract group patients completed the CDT in the specific picture manner (the short hand on 11 and the long hand between 11 and 12), (P = 0.001). Conclusions: Lower CDT results were seen in NTG patients according to two different scoring systems. NTG patients showed a specific manner of drawing. Further prospective studies are needed to investigate the CDT reliability as fast screening test of cognitive function impairment in glaucoma patients

Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the microenvironment on globin gene expression. Adult progenitors express high levels of c-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, c-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in c-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and c-globin promoter DNA methylation. We conclude that c-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

Effect of AFT024 and U26B1 cell lines on γ-globin expression requires cell-cell contact.

<p>Analysis of γ-globin chain synthesis (γ/γ+β) in baboon BM-derived CD34+ cells cultured in methylcellulose (MC), liquid media (LM), and AFT024 and U26 B1 co-cultures, transwell (TW) cultures and in LM containing conditioned media (CM) from the AFT024 and U26B1 cell lines. The single asterisk denotes statistical significance (p<.01) relative to AFT co-cultures. The double asterisk denotes statistical significance (p<.01) relative to U26B1 co-cultures.</p

Roles of DNA methylation and development in modulation of γ-globin expression in AFT024 co-cultures. A.

<p>Bisulfite sequence analysis of DNA methylation of the γ-globin 5′ promoter region in erythroid progenitors grown in methylcellulose media, liquid media, or in co-cultures containing the AFT024 stromal cell line. Five CpG sites numbered relative to the transcription start site were analyzed. Each row represents the sequence analysis of a single amplicon cloned within the pCR4 vector. Unmethylated sites (green), methylated site (red), polymorophic sites where no CpG is present (yellow). <b>B.</b> Effect of decitabine on globin chain synthesis (γ/γ+β) during erythroid differentiation of baboon BM progenitors in AFT024 co-cultures. Decitabine (5×10⁻⁷ M) was added to cultures on d7. Globin chain synthesis was analyzed on d11 and d14. <b>C.</b> Globin chain synthesis (γ/γ+β) during erythroid differentiation of human cord blood progenitors in AFT024 co-cultures.</p

Effect of stromal cell line co-cultures on γ-globin expression during erythroid differentiation of CD34+ bone marrow progenitors.

<p><b>A.</b> Comparison of globin chain synthesis on varying days in baboon BM-derived erythroid progenitors grown in liquid media and in AFT024 stromal cell line co-cultures. <b>B.</b> Globin chain synthesis in baboon BM-derived erythroid progenitors grown in methylcellulose media (MC), liquid media (LM), and co-cultures containing the AFT024, U26B1, or OP9 stromal cell lines. <b>C.</b> Comparison of globin mRNA expression (γ/γ+β) in baboon BM-derived erythroid progenitors grown in methylcellulose media, liquid media, or AFT024 stromal cell line co-cultures. <b>D.</b> Globin chain synthesis in CD34+ baboon BM subpopulations differing in CD36 expression.</p