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Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.

Hajizadeh-Saffar E, Tahamtani Y, Aghdami N, Azadmanesh K, Habibi-Anbouhi M, Heremans Y, De Leu N, Heimberg H, Ravassard P, Shokrgozar MA, Baharvand H - Sci Rep (2015)

Bottom Line: Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF.Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone.We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice.

View Article: PubMed Central - PubMed

Affiliation: 1] National Cell Bank, Pasteur Institute of Iran, Tehran, Iran [2] Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

ABSTRACT

Unlabelled: Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF.

Hesc-msc: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.

No MeSH data available.


Related in: MedlinePlus

Lentiviral transduction of hESC-MSCs for inducible expression of VEGF.(a) Phase contrast (left) and fluorescent (right) images of double transduced hESC-MSCs at passage 3. (b) VEGF ELISA for transduced hESC-MSCs (hESC-MSC:VEGF) in +Dox (passages 3 and 5) or -Dox. (c) Tube formation assay (HUVECs stained by Calcein AM) and quantification by Image J, calculating the numbers of tubes (d) branch points (e) and tube area per field (f); five fields per sample, n = 3. (g) Glucose stimulated insulin secretion in 2.8, 8.3, 16.7 and 22 mM glucose and (h) Stimulation index (ratio of highly stimulated over basal insulin secretion) of islets co-cultured with hESC-MSC:VEGF. Values represent mean ± SD, ***p < 0.005. Dox: Doxycycline.
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f2: Lentiviral transduction of hESC-MSCs for inducible expression of VEGF.(a) Phase contrast (left) and fluorescent (right) images of double transduced hESC-MSCs at passage 3. (b) VEGF ELISA for transduced hESC-MSCs (hESC-MSC:VEGF) in +Dox (passages 3 and 5) or -Dox. (c) Tube formation assay (HUVECs stained by Calcein AM) and quantification by Image J, calculating the numbers of tubes (d) branch points (e) and tube area per field (f); five fields per sample, n = 3. (g) Glucose stimulated insulin secretion in 2.8, 8.3, 16.7 and 22 mM glucose and (h) Stimulation index (ratio of highly stimulated over basal insulin secretion) of islets co-cultured with hESC-MSC:VEGF. Values represent mean ± SD, ***p < 0.005. Dox: Doxycycline.

Mentions: HESC-MSCs were transduced with rtTA- and TetO-VEGF-expressing lentiviruses at three different multiplicities of infection (MOIs; 5, 25 and 50). Since both lentiviruses constitutively expressed GFP, we determined the percentage of transduced cells by flow cytometry. The results showed significantly higher transduction rate with Le-rtTA at all MOIs (82 ± 9.7% at MOI 50) compared to Le-TetO-VEGF lentivirus (38 ± 9% at MOI 50; Supplementary Figure S2-d online). Therefore, VEGF-transduced hESC-MSCs were sorted (Supplementary Figure S2-e online) and subsequently transduced with Le-rtTA. The resulting cells showed normal morphology and expansion potential and were used for co-transplantation experiments when they reached passage three (Figure 2a).


Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.

Hajizadeh-Saffar E, Tahamtani Y, Aghdami N, Azadmanesh K, Habibi-Anbouhi M, Heremans Y, De Leu N, Heimberg H, Ravassard P, Shokrgozar MA, Baharvand H - Sci Rep (2015)

Lentiviral transduction of hESC-MSCs for inducible expression of VEGF.(a) Phase contrast (left) and fluorescent (right) images of double transduced hESC-MSCs at passage 3. (b) VEGF ELISA for transduced hESC-MSCs (hESC-MSC:VEGF) in +Dox (passages 3 and 5) or -Dox. (c) Tube formation assay (HUVECs stained by Calcein AM) and quantification by Image J, calculating the numbers of tubes (d) branch points (e) and tube area per field (f); five fields per sample, n = 3. (g) Glucose stimulated insulin secretion in 2.8, 8.3, 16.7 and 22 mM glucose and (h) Stimulation index (ratio of highly stimulated over basal insulin secretion) of islets co-cultured with hESC-MSC:VEGF. Values represent mean ± SD, ***p < 0.005. Dox: Doxycycline.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4377549&req=5

f2: Lentiviral transduction of hESC-MSCs for inducible expression of VEGF.(a) Phase contrast (left) and fluorescent (right) images of double transduced hESC-MSCs at passage 3. (b) VEGF ELISA for transduced hESC-MSCs (hESC-MSC:VEGF) in +Dox (passages 3 and 5) or -Dox. (c) Tube formation assay (HUVECs stained by Calcein AM) and quantification by Image J, calculating the numbers of tubes (d) branch points (e) and tube area per field (f); five fields per sample, n = 3. (g) Glucose stimulated insulin secretion in 2.8, 8.3, 16.7 and 22 mM glucose and (h) Stimulation index (ratio of highly stimulated over basal insulin secretion) of islets co-cultured with hESC-MSC:VEGF. Values represent mean ± SD, ***p < 0.005. Dox: Doxycycline.
Mentions: HESC-MSCs were transduced with rtTA- and TetO-VEGF-expressing lentiviruses at three different multiplicities of infection (MOIs; 5, 25 and 50). Since both lentiviruses constitutively expressed GFP, we determined the percentage of transduced cells by flow cytometry. The results showed significantly higher transduction rate with Le-rtTA at all MOIs (82 ± 9.7% at MOI 50) compared to Le-TetO-VEGF lentivirus (38 ± 9% at MOI 50; Supplementary Figure S2-d online). Therefore, VEGF-transduced hESC-MSCs were sorted (Supplementary Figure S2-e online) and subsequently transduced with Le-rtTA. The resulting cells showed normal morphology and expansion potential and were used for co-transplantation experiments when they reached passage three (Figure 2a).

Bottom Line: Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF.Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone.We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice.

View Article: PubMed Central - PubMed

Affiliation: 1] National Cell Bank, Pasteur Institute of Iran, Tehran, Iran [2] Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

ABSTRACT

Unlabelled: Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF.

Hesc-msc: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.

No MeSH data available.


Related in: MedlinePlus