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Human Bone Marrow Subpopulations Sustain Human Islet Function and Viability In vitro.

Luo LG, Xiong F, Ravassard P, Luo JZ - Br J Med Med Res (2015)

Bottom Line: However, only whole BM enables to sustain the capability of islet β-cell self regeneration resulting in increasing β cell population while single E and M individual do not significantly affect on that.Mechanism approach to explore β-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of β-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a).These results suggest that BM and its derived M and E cells enable to support human islet β-cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine/Research, Roger Williams Medical Center, Boston University, USA.

ABSTRACT

Aims: Allogeneic bone marrow (BM) has been shown to support human islet survival and function in long-term culture by initiating human islet vascularization and β-cell regeneration. Various BM subpopulations may play different roles in human islet functions and survival. In this paper we investigated the effects of BM and its subpopulations, endothelial progenitor cells (E) and mesenchymal (M) cells on human islet's β-cell function and regeneration.

Study design: Isolation and identification of subpopulations from human bone marrow and culture with allogeneic human islet to investigate effects of different cell population on human islet function and regeneration.

Place and duration of study: Department of Medicine, Center for Stem Cell & Diabetes Research, RWMC, Providence, RI, USA, between 2010 - 2014.

Methodology: Human islets were distributed from Integrated Islet Distribution Program (IIDP) and human bone marrow (BM) was harvested by Bone marrow transplantation center at Roger Williams Hospital. BM subpopulation was identified cell surface markers through Fluorescence-activated cell sorting, applied in flow cytometry (FACS), islet function was evaluated by human ELISA kit and β cell regeneration was evaluated by three methods of Cre-Loxp cell tracing, β cell sorting and RT-PCR for gene expression.

Results: Four different BM and seven different islet donates contributed human tissues. We observed islet β-cell having self regeneration capability in short term culture (3∼5 days) using a Cre-Loxp cell tracing. BM and its subtype E, M have similar benefits on β cell function during co-culture with human islet comparison to islet only. However, only whole BM enables to sustain the capability of islet β-cell self regeneration resulting in increasing β cell population while single E and M individual do not significantly affect on that. Mechanism approach to explore β-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of β-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a).

Conclusion: These results suggest that BM and its derived M and E cells enable to support human islet β-cell function. However, only BM can sustain the capability of β-cell self regeneration through initiating β-cell transcriptional factors but not individual E and M cells suggesting pure E and M cells less supportive for islet long-term survival in vitro.

No MeSH data available.


Related in: MedlinePlus

mwBM enhances β-cell regeneration in vitro but not E and MCultured human islet cells labeled by Cre-loxp with mwBM, E and M were monitored under microscope with quantification of accounting green positive cells during culture days of 6, 10, 19 and 28 (Fig. 3 A). Top image panels show four groups Cre-Loxp positive cells in human islet culture, bottom panel indicates cre-loxp green cells quantification. Flow cytometry quantification method to quantify total positive insulin cells change after 28 days culture with different BM cell populations (Fig. 3 B)
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Figure 3: mwBM enhances β-cell regeneration in vitro but not E and MCultured human islet cells labeled by Cre-loxp with mwBM, E and M were monitored under microscope with quantification of accounting green positive cells during culture days of 6, 10, 19 and 28 (Fig. 3 A). Top image panels show four groups Cre-Loxp positive cells in human islet culture, bottom panel indicates cre-loxp green cells quantification. Flow cytometry quantification method to quantify total positive insulin cells change after 28 days culture with different BM cell populations (Fig. 3 B)

Mentions: During the cultures, effects of M, E and mwBM on human islet β-cell population was monitored by counting labeled human islet with Cre-Loxp on Days 6, 10, 19 and 28. As shown by previous data (Fig. 3), no significant differences in insulin positive cell population by cell counting represents amongst the four groups of culture in the first four days. MwBM co-culture showed a significant increase in insulin positive cells until day 28 (p< 0.01). M and E had no significant effects in terms of increase counting insulin positive cells (P< 0.05) (Figs. 3 a, b). Utilizing cell quantification via a two-step Flow Cytometry method to further quantify the total number of insulin positive cells, the results were consistent with Cre-Loxp studies. MwBM significantly increased human islet β-cell numbers throughout 4 weeks of culture. This effect was not seen in M and E cocultured islet (Fig. 3 c).


Human Bone Marrow Subpopulations Sustain Human Islet Function and Viability In vitro.

Luo LG, Xiong F, Ravassard P, Luo JZ - Br J Med Med Res (2015)

mwBM enhances β-cell regeneration in vitro but not E and MCultured human islet cells labeled by Cre-loxp with mwBM, E and M were monitored under microscope with quantification of accounting green positive cells during culture days of 6, 10, 19 and 28 (Fig. 3 A). Top image panels show four groups Cre-Loxp positive cells in human islet culture, bottom panel indicates cre-loxp green cells quantification. Flow cytometry quantification method to quantify total positive insulin cells change after 28 days culture with different BM cell populations (Fig. 3 B)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837454&req=5

Figure 3: mwBM enhances β-cell regeneration in vitro but not E and MCultured human islet cells labeled by Cre-loxp with mwBM, E and M were monitored under microscope with quantification of accounting green positive cells during culture days of 6, 10, 19 and 28 (Fig. 3 A). Top image panels show four groups Cre-Loxp positive cells in human islet culture, bottom panel indicates cre-loxp green cells quantification. Flow cytometry quantification method to quantify total positive insulin cells change after 28 days culture with different BM cell populations (Fig. 3 B)
Mentions: During the cultures, effects of M, E and mwBM on human islet β-cell population was monitored by counting labeled human islet with Cre-Loxp on Days 6, 10, 19 and 28. As shown by previous data (Fig. 3), no significant differences in insulin positive cell population by cell counting represents amongst the four groups of culture in the first four days. MwBM co-culture showed a significant increase in insulin positive cells until day 28 (p< 0.01). M and E had no significant effects in terms of increase counting insulin positive cells (P< 0.05) (Figs. 3 a, b). Utilizing cell quantification via a two-step Flow Cytometry method to further quantify the total number of insulin positive cells, the results were consistent with Cre-Loxp studies. MwBM significantly increased human islet β-cell numbers throughout 4 weeks of culture. This effect was not seen in M and E cocultured islet (Fig. 3 c).

Bottom Line: However, only whole BM enables to sustain the capability of islet β-cell self regeneration resulting in increasing β cell population while single E and M individual do not significantly affect on that.Mechanism approach to explore β-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of β-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a).These results suggest that BM and its derived M and E cells enable to support human islet β-cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine/Research, Roger Williams Medical Center, Boston University, USA.

ABSTRACT

Aims: Allogeneic bone marrow (BM) has been shown to support human islet survival and function in long-term culture by initiating human islet vascularization and β-cell regeneration. Various BM subpopulations may play different roles in human islet functions and survival. In this paper we investigated the effects of BM and its subpopulations, endothelial progenitor cells (E) and mesenchymal (M) cells on human islet's β-cell function and regeneration.

Study design: Isolation and identification of subpopulations from human bone marrow and culture with allogeneic human islet to investigate effects of different cell population on human islet function and regeneration.

Place and duration of study: Department of Medicine, Center for Stem Cell & Diabetes Research, RWMC, Providence, RI, USA, between 2010 - 2014.

Methodology: Human islets were distributed from Integrated Islet Distribution Program (IIDP) and human bone marrow (BM) was harvested by Bone marrow transplantation center at Roger Williams Hospital. BM subpopulation was identified cell surface markers through Fluorescence-activated cell sorting, applied in flow cytometry (FACS), islet function was evaluated by human ELISA kit and β cell regeneration was evaluated by three methods of Cre-Loxp cell tracing, β cell sorting and RT-PCR for gene expression.

Results: Four different BM and seven different islet donates contributed human tissues. We observed islet β-cell having self regeneration capability in short term culture (3∼5 days) using a Cre-Loxp cell tracing. BM and its subtype E, M have similar benefits on β cell function during co-culture with human islet comparison to islet only. However, only whole BM enables to sustain the capability of islet β-cell self regeneration resulting in increasing β cell population while single E and M individual do not significantly affect on that. Mechanism approach to explore β-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of β-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a).

Conclusion: These results suggest that BM and its derived M and E cells enable to support human islet β-cell function. However, only BM can sustain the capability of β-cell self regeneration through initiating β-cell transcriptional factors but not individual E and M cells suggesting pure E and M cells less supportive for islet long-term survival in vitro.

No MeSH data available.


Related in: MedlinePlus