Research ArticleISLET TRANSPLANTATION

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization

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Science Translational Medicine  15 Jan 2020:
Vol. 12, Issue 526, eaan5907
DOI: 10.1126/scitranslmed.aan5907
  • Fig. 1 HUCPVCs grown in different GMP-compliant media.

    (A) Representative phenotypic analysis of HUCPVCs grown in GMP-compliant medium (SMAB). HUCPVCs p3, baseline fluorescence set using matched isotype controls. Right: Percentage of HUCPVCs expressing a CD14CD45CD73+CD90+CD105+ phenotype across three serial passages (p2 to p4). n = 3 individual donations. (B) HUCPVCs grown in different GMP media across three passages (p2 to p4) from n = 5 donated samples of umbilical cord. Mean doubling time ± SEM (days) using different GMP-compliant media. No significant differences between media were used to expand cell lines with two-way ANOVA with Tukey’s multiple comparison test. P ranges from 0.22 to 0.99. (C) Colony-forming unit scores for HUCPVCs and lipoaspirate MSCs grown in different GMP-compliant media from three different tissue donations across three passages (p2 to p4) with numbers of colonies obtained from plating 10 cells/cm2 in CFU-F medium; quantified mean ± SEM is shown; CFU-F is significantly higher in HUCPVC than in lipoaspirate cells (P < 0.01), paired t test in all media tested. (D) Differentiation of HUCPVCs to adipocytes, osteocytes, and chondrocytes. Samples were stained with Oil Red O (Adipose), Alizarin Red (Osteo), or Safranin O (Chondro) as described (63). Bright-field microscopy magnification, ×40. (E) Analysis of PD-L2 (CD273) and PD-L1 (CD274) expression on HUCPVCs with and without licensing. Light gray peak/green outline, isotype control; gray peak/blue outline, unlicensed; white peak, licensed. SM, StemMACS; SMAB, SM + AB plasma; SMPL, SM + platelet lysate; CFU-F, colony-forming unit–fibroblasts.

  • Fig. 2 Inhibition of T cell proliferation and requirement for IFN-γ licensing for HUCPVCs to mediate this effect.

    (A) Representative dye dilution results measuring inhibition of T cell proliferation by HUCPVCs. HUCPVCs were grown in SMAB, assayed at p4, and cultured with Ef670-stained PBMCs; ratios are PBMC:HUCPVC at outset of culture. PHA, phytohemagglutinin. (B) Inhibition of T cell proliferation with and without IFN-γ licensing. Comparative measurement of inhibitory effects was made by comparing the percentage of T cells remaining in the undivided peak after activation with or without titrated amounts of MSCs. Mean ± SD of three different HUCPVC lines grown in SMPL ratios are PBMC:HUCPVC (no significant differences between licensed and unlicensed groups at each concentration, paired t test).

  • Fig. 3 Gene expression and protein production by resting and licensed MSCs.

    (A) TGF-β, (B) CD274, (C) IDO, and (D) TSG-6 gene expression in resting and licensed MSCs from lipoaspirate (blue) and HUCPVCs (red), measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed as 2(−ΔCT) against expression of RPLP0. Means ± SEM (n = 3 donors), MSC at p3. Where plain bars are absent, gene expression had lower limit of detection and was not included in measurement of significance. (E) CXCL8 and (F) VEGF production by resting (plain bars) and licensed (hatched bars) MSCs from lipoaspirate (blue) and HUCPVCs (red) measured by Luminex 100 (in pg/ml). Means ± SEM (n = 3 donors), HUCPVCs and MSCs at p3. Protein measurements showed net of background measurement of analyte in culture medium alone.

  • Fig. 4 Chemokine expression by resting and licensed lipo-MSCs and HUCPVC.

    The expression of inflammatory, ELR+ve, and ELR−ve chemokines by resting (−) and licensed (+) MSCs from lipo-MSC (adipose) and HUCPVCs (umbilical cord) were measured by RT2 Profiler PCR Arrays. The expression of anti-inflammatory genes was measured using qRT-PCR as indicated. All samples were run at the same time on the same plate. In each case, the average 2(−ΔCT) is plotted, and heat maps were generated using Heatmapper software (www2.heatmapper.ca/). Each group of genes was analyzed separately. Genes are clustered using the single linkage method. n > 3 < 6 donors, HUCPVC/MSCs at p3 (expression of CD274, TSG-6, IDO, and TGF-β same samples as Fig. 3A).

  • Fig. 5 Time to cure diabetes with IPGTTs in NSG mice receiving 3000 IEQs and HUCPVCs.

    (A) All mice were transplanted with 3000 IEQs plus different ratios of IEQ:HUCPVCs (1:0, 1:10, 1:30, and 1:50). Time to normoglycemia (nonfasted glucose <11.0 mM) was determined. Tail vein sampling for glucose concentrations (mM) during IPGTT (2 g/kg) was done at 0, 15, 30, 60, 90, and 120 min after IPGTT. Stimulated C-peptide concentrations (pM) were sampled at 60 min after IPGTT. Glucose concentrations at (B) 2.8 weeks, (C) 7 weeks, (D) 12 weeks, and (E) 16 weeks after islet transplant. Stimulated C-peptide concentration (pM) divided by glucose concentration (mM) at (F) 12 weeks and (G) 16 weeks. n = 6 to 8 mice per group. Data presented as mean ± SEM. See Table 1 for additional information. Black bar, 3000 IEQs alone; red bar, 1:10 ratio of IEQ:HUCPVCs; red horizontal lined bar, 1:30 ratio of IEQ:HUCPVCs; red vertical lined bar, 1:50 ratio of IEQ:HUCPVCs. Post hoc analyses for significant differences between ratios of IEQ:HUCPVCs are denoted: a, 1:0 versus 1:10; b, 1:0 versus 1:30; c, 1:0 versus 1:50; d, 1:10 versus 1:30; e, 1:10 versus 1:50; f, 1:30 versus 1:50.

  • Fig. 6 Vessel density in islet grafts implanted under kidney capsule at 16 weeks after transplant.

    Diabetic NSG mice were transplanted with 3000 IEQs ± HUCPVCs (IEQ:HUCPVC ratios of 1:0, 1:10, 1:30, and 1:50). Serial sections (5 μm) in n = 5 mice per group were cut through the entire islet graft below the kidney capsule, and vessel density associated with the islet graft was quantified. (A) Vessel density at 16 weeks after transplant; data presented as mean ± SEM. Black bar, 3000 IEQs alone; red bar, 1:10 ratio of IEQ:HUCPVCs; red horizontal lined bar, 1:30 ratio of IEQ:HUCPVCs; red vertical lined bar, 1:50 ratio of IEQ:HUCPVCs. Post hoc analyses for significant differences between ratios of IEQ:HUCPVCs are denoted: a, 1:0 versus 1:10; b, 1:0 versus 1:30; c, 1:0 versus 1:50; d, 1:10 versus 1:30; e, 1:10 versus 1:50; f, 1:30 versus 1:50. (B) Immunofluorescence image of a representative section showing vessel quantification in an islet graft. Endothelial cells (isolectin B4) and mature vessels (anti–smooth muscle α-actin antibody) were identified by immunostaining. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 7 Blood glucose concentrations after islet transplant ± HUCPVCs in mismatch (allogeneic) and syngeneic transplants.

    (A) Immunocompetent C57Bl/6 mice (n = 4 to 6 per group) were transplanted with allogeneic BALB/c islets (n = 500 IEQs) and HUCPVCs (ratios, 1:0, 1:30, 1:90, and 1:150). Mean values of glucose concentration are shown (P > 0.05). (B) Glucose concentration over time, comparing islet-only grafts (n = 900 IEQs) to IEQ:HUCPVCs (1:150) in immunocompetent C57Bl/6 mice transplanted with BALB/c islets (n = 8 mice per group). (C) Immunocompetent C57Bl/6 mice (n = 8 per group) were transplanted with syngeneic C57Bl/6 mouse islets and HUCPVCs (ratios, 1:0, 1:90, 1:150, and 1:210). Mean values of glucose concentration are shown. Tx, transplant. (D) Stimulated (60 min) insulin concentration (pM) after IPGTT divided by glucose concentration (mM) at 6 weeks in immunocompetent syngeneic islet transplants. n = 8 mice per group. Data are presented as mean ± SEM.

  • Fig. 8 Blood glucose measurement after human islet ± HUCPVC transplant in immunodeficient mice.

    Immunodeficient NSG mice (n = 8 per group) were transplanted with 700 human islets ± HUCPVCs in a ratio of 1:150 IEQ:HUCPVCs. Mean ± SEM values are shown; difference between groups, P < 0.01.

  • Table 1 AUC of glucose concentrations after IPGTT.

    Data presented as mean ± SEM. The integrated area under the curve of glucose concentrations was calculated during the 120-min IPGTT (mM × min2) at 2.8, 7, 12, and 16 weeks after islet transplant; n = 6 to 8 mice per group. One-way ANOVA analyses comparing effect of integrated glucose response with ratio of islets: HUCPVCs transplanted under the kidney capsule were compared at separate time intervals. Post hoc analyses for significant differences between ratios of islets to HUCPVCs are denoted: a, 1:0 versus 1:10; b, 1:0 versus 1:30; c, 1:0 versus 1:50; d, 1:10 versus 1:30; e, 1:10 versus 1:50; f, 1:30 versus 1:50.

    Weeks after transplantIslet:HUCPVC 1:0Islet:HUCPVC 1:10Islet:HUCPVC 1:30Islet:HUCPVC 1:50P ANOVA
    2.8 weeks970 ± 8430 ± 20457 ± 9457 ± 23<0.0001a,b,c
    7 weeks902 ± 171672 ± 35473 ± 15691 ± 530.02b
    12 weeks1111 ± 1231218 ± 40418 ± 34441 ± 30<0.0001b,c,d,e
    16 weeks1089 ± 601045 ± 58544 ± 29721 ± 19<0.0001b,c,d,e,f

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/526/eaan5907/DC1

    Materials and Methods

    Fig. S1. Characterization of lipoaspirate-derived MSCs in vitro.

    Fig. S2. Inhibition of T cell proliferation by HUCPVC.

    Fig. S3. Characterization of lipoaspirate-derived MSCs in vivo.

    Fig. S4. AUC glucose 6 weeks after transplant.

    Fig. S5. Glucose concentrations after removal of kidney islet graft by nephrectomy.

    Fig. S6. Immunofluorescence analysis with endothelial marker ERG.

    Fig. S7. Vessel density in islet grafts with and without HUCPVCs.

    Fig. S8. Histological assessment of cell infiltrate into liver after syngeneic islet grafts ± HUCPVC.

    Fig. S9. Liver function 6 weeks after transplant.

    Table S1. Human islet preparation and associated clinical characteristics.

    Table S2. GSIS and OCR of islet preparations.

    Table S3. Glucose concentrations and weights before surgery.

    Data file S1. Primary data.

    References (6471)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of lipoaspirate-derived MSCs in vitro.
    • Fig. S2. Inhibition of T cell proliferation by HUCPVC.
    • Fig. S3. Characterization of lipoaspirate-derived MSCs in vivo.
    • Fig. S4. AUC glucose 6 weeks after transplant.
    • Fig. S5. Glucose concentrations after removal of kidney islet graft by nephrectomy.
    • Fig. S6. Immunofluorescence analysis with endothelial marker ERG.
    • Fig. S7. Vessel density in islet grafts with and without HUCPVCs.
    • Fig. S8. Histological assessment of cell infiltrate into liver after syngeneic islet grafts ± HUCPVC.
    • Fig. S9. Liver function 6 weeks after transplant.
    • Table S1. Human islet preparation and associated clinical characteristics.
    • Table S2. GSIS and OCR of islet preparations.
    • Table S3. Glucose concentrations and weights before surgery.
    • References (6471)

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    Other Supplementary Material for this manuscript includes the following:

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