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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

Isolation and identification of E (EPCs) and M (MSCs)Attached M in culture (Fig. 1 A left up image day 1) after one week culture (Fig. 1 A left bottom day 7) were scribed by rubber scriber and labeled with cell surface maker CD-73 and 90 after flow cytometry analysis (Fig. 1 A right panels) shows more than 95% cells are CD-73 and 90 positive cells. Cultured one week E cells (Fig. 1 B left image) were released and labeled with cell surface markers CD-135, 90, 31 and 34. Flow cytometry analysis indicated that these cells include most of positive CD-135, 90 and 31 but bare 34 positive. (Fig. 1 B right panels)
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Figure 1: Isolation and identification of E (EPCs) and M (MSCs)Attached M in culture (Fig. 1 A left up image day 1) after one week culture (Fig. 1 A left bottom day 7) were scribed by rubber scriber and labeled with cell surface maker CD-73 and 90 after flow cytometry analysis (Fig. 1 A right panels) shows more than 95% cells are CD-73 and 90 positive cells. Cultured one week E cells (Fig. 1 B left image) were released and labeled with cell surface markers CD-135, 90, 31 and 34. Flow cytometry analysis indicated that these cells include most of positive CD-135, 90 and 31 but bare 34 positive. (Fig. 1 B right panels)

Mentions: Bone marrow mesenchymal cell isolation and identification: Briefly, after three days of culture, floating cells were aspirated leaving only the attached cells for continued culture which include most of mesenchymal cells (M). Culture for three days, detached cells from attached culture and cells were labeled with CD73 and CD90 antibodies and identified by flow cytometry. The results indicated that >95% cells were either CD73 or CD90 positive suggesting that these isolated cells from BM were M. Using same technique, we characterize cell populations from cultures with CD31, CD73, CD135 and CD 34 antibodies and identified by flow cytometry. The results indicated that > 14% cells were CD31, CD73, CD135 positive but less than 1.5% was CD34 positive, suggesting that these isolated cells from BM were endothelial cells (E) (Fig. 1).


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)

Isolation and identification of E (EPCs) and M (MSCs)Attached M in culture (Fig. 1 A left up image day 1) after one week culture (Fig. 1 A left bottom day 7) were scribed by rubber scriber and labeled with cell surface maker CD-73 and 90 after flow cytometry analysis (Fig. 1 A right panels) shows more than 95% cells are CD-73 and 90 positive cells. Cultured one week E cells (Fig. 1 B left image) were released and labeled with cell surface markers CD-135, 90, 31 and 34. Flow cytometry analysis indicated that these cells include most of positive CD-135, 90 and 31 but bare 34 positive. (Fig. 1 B right panels)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Isolation and identification of E (EPCs) and M (MSCs)Attached M in culture (Fig. 1 A left up image day 1) after one week culture (Fig. 1 A left bottom day 7) were scribed by rubber scriber and labeled with cell surface maker CD-73 and 90 after flow cytometry analysis (Fig. 1 A right panels) shows more than 95% cells are CD-73 and 90 positive cells. Cultured one week E cells (Fig. 1 B left image) were released and labeled with cell surface markers CD-135, 90, 31 and 34. Flow cytometry analysis indicated that these cells include most of positive CD-135, 90 and 31 but bare 34 positive. (Fig. 1 B right panels)
Mentions: Bone marrow mesenchymal cell isolation and identification: Briefly, after three days of culture, floating cells were aspirated leaving only the attached cells for continued culture which include most of mesenchymal cells (M). Culture for three days, detached cells from attached culture and cells were labeled with CD73 and CD90 antibodies and identified by flow cytometry. The results indicated that >95% cells were either CD73 or CD90 positive suggesting that these isolated cells from BM were M. Using same technique, we characterize cell populations from cultures with CD31, CD73, CD135 and CD 34 antibodies and identified by flow cytometry. The results indicated that > 14% cells were CD31, CD73, CD135 positive but less than 1.5% was CD34 positive, suggesting that these isolated cells from BM were endothelial cells (E) (Fig. 1).

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