Highly Angiogenic, Nonthrombogenic Bone Marrow Mononuclear Cell–Derived Spheroids in Intraportal Islet Transplantation
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Figures
- Figure 1
Histological assessment of liver tissue that received transplanted MSCs, MSC-spheroids, and BM-spheroids. A: Approximately 300 MSC-spheroids (containing 3 × 105 cells [∼1 × 103 cells per spheroid]), ∼100 MSC-spheroids (containing 1 × 106 cells [∼1 × 104 cells per spheroid]), and MSC-monolayers (1 × 105, 2.5 × 105, and 5 × 105) were transplanted via the portal vein. After 24 h, each liver was extracted and observed for the presence of infarcted areas. Black arrows indicate the infarcted areas. B: Approximately 150 BM-spheroids (containing 5 × 105 cells), 300 BM-spheroids (containing 1 × 106 cells), 600 BM-spheroids (containing 2 × 106 cells), and dissociated BM-spheroids (5 × 105 and 1 × 106 cells) were intraportally injected. Each liver was examined for the presence of infarcted areas. C: MSC-monolayers, MSC-spheroids, and BM-spheroids were transplanted via the portal vein. At DPT 1, liver tissues were prepared for H-E staining. Livers that received intraportally transplanted MSCs showed pathological changes, but those that received BM-spheroids did not. Arrows indicate infarction regions. Scale bars, 2 mm. Enlarged regions from the black square boxes indicate the infiltration of immune cells. Scale bars, 100 μm. D: GFP MSC-monolayers and -spheroids were used to trace the behavior of MSC-monolayers and MSC-spheroids after intraportal injection. Liver tissues were stained with CD31 (red) for vessels and GFP (green) for MSCs. The asterisk indicates necrotic area, and the white arrow indicates blood clot formation. Scale bars, 100 μm.
- Figure 2
Coagulatory and inflammatory profiles of MSCs, MSC-spheroids, and BM-spheroids by microarray analysis and Western blot assay. A: Microarray analysis of the relative gene expression levels of BM-spheroids (test) and MSC-spheroids (control). Genes with P < 0.05 and more than fourfold change in expression in three independent experiments were used to create the heat map, which was based on coagulatory and inflammatory gene expression profiles in BM-spheroids vs. MSC-spheroids. Red and green in the heat map indicate up- and downregulation in BM-spheroids compared with MSC-spheroids, respectively. B: Representative images of TF staining of intraportally injected GFP MSC-monolayers, MSC-spheroids, and BM-spheroids. Infused cells were stained with TF (red) and GFP (green). Scale bars, 100 μm. C and D: Western blot analysis of TF and CCL2 protein levels was performed for MSC-monolayers, MSC-spheroids of different sizes (1 × 103 cells per spheroid, 2.5 × 103 cells per spheroid, 5 × 103 cells per spheroid, and 1 × 104 cells per spheroid), BM-MNCs, and BM-spheroids. C: Representative immunoblotting. D: Quantitative analysis of band intensity. *P < 0.05; ***P < 0.001. The data are depicted as mean ± SEM (n = 3) values and normalized by β-actin. Max, maximum; Min, minimum.
- Figure 3
Reversal of diabetes after intraportal cotransplantation of BM-spheroids and islets. A: Experimental scheme of intraportal cotransplantation of islets and BM-spheroids. Experimental animals were divided into three groups: islets alone (300 handpicked islets), islets (300 islets) plus 150–200 BM-spheroids, and islets (300 islets) plus 5 × 105 single cells dissociated from BM-spheroids. Islets and BM-spheroids were cotransplanted through the portal vein of mice with STZ-induced diabetes. B–D: Blood glucose concentration of mice intraportally transplanted with islets alone (n = 15) (islets alone group) (B), islets plus BM-spheroids (n = 12) (BM-spheroid group) (D), or islets plus single cells dissociated from BM-spheroids (n = 8) (dissociated BM-spheroid group) (C) was measured up to DPT 28. The mean blood glucose level was 399 ± 52 mg/dL in islets alone, 375 ± 69 mg/dL in dissociated BM-spheroid, and 184 ± 23 mg/dL in BM-spheroid at DPT 28. E: Cumulative diabetes reversal curve after islet transplantation. Normoglycemia was defined as <200 mg/dL on consecutive days. *P < 0.05 for log-rank test of islets alone (4/15) or dissociated BM-spheroid (3/8) vs. BM-spheroid (12/14). F: Glucose tolerance test at DPT 28. Blood glucose levels were measured at 0, 15, 30, 45, 60, 90, and 120 min after glucose (1 g/kg) injection. G: Area under the curve (AUCglu) for intraperitoneal glucose tolerance test. *P < 0.05. H: Fasting serum insulin levels at 0 and 30 min after glucose loading. *P < 0.05, one-way ANOVA followed by Tukey multiple comparisons test.
- Figure 4
Assessment of morphology and revascularization for graft-bearing liver cotransplanted with BM-spheroids. A: At DPT 28, the graft-bearing liver tissues were stained for insulin. Representative images of islets transplanted via the portal vein are shown. Regions denoted by a black line indicate magnified images (right panel). Scale bars, 50 μm. B–D: Evaluation of fractional β-cell area (B), islet size (C), and islet number/total area (D) for the grafted tissues of each group. *P < 0.05; ***P < 0.001. E: For tracing of the transplanted BM-spheroids and functional vessels, GFP BM-spheroids derived from GFP-Tg mice were cotransplanted with islets, and rhodamine-labeled BS1 lectin was intracardially injected. Liver tissues were stained with GFP antibody to determine how many GFP cells were present at DPT 14. Regions denoted by a white line indicate magnified images (right panel). Scale bars of left panel, 1 mm, and scale bars of right panel, 100 μm. F: At DPT 14 and 28, representative images indicate vessel formation at the graft site. Vessels, islets, and GFP BM-spheroids were stained for lectin (red), insulin (blue), and GFP (green). Scale bars, 100 μm. G and H: Quantification of the vessel density of each group at DPT 14 (G) and DPT 28 (H). *P < 0.05, one-way ANOVA followed by Tukey multiple comparisons test. ECs, endothelial cells.
- Figure 5
Tracing of transplanted BM-spheroids. A: Bioluminescent imaging. GFP BM-spheroids and GFP dissociated BM-spheroids were intraportally transplanted. Mice of each group were injected with the same number of GFP cells (1 × 106 cells). Liver, spleen, and lung were imaged at DPT 1. Control is the liver without transplanted GFP BM-spheroids. The single bar indicates relative photon light intensity. B: Cell ex vivo MRI. The cell imaging of BM-spheroids labeled with ferucarbotran was performed by MRI. The left panel is a BM-spheroid image obtained by optical microscopy, and the right panel is visible hypointense spots representing ferucarbotran-labeled BM-spheroids. Scale bar, 100 μm. C: Liver MRI images of ferucarbotran-labeled BM-spheroids and dissociated BM-spheroids transplanted via the portal vein. MRI images were obtained at DPT 1 and 8. Control is liver MRI images without transplanted BM-spheroids. White elliptical regions are distinctly visible hypointense spots representing ferucarbotran-labeled BM-spheroids and dissociated BM-spheroids. Enlarged regions from white elliptical regions of the middle panel are shown in the right panel. D: IVM for monitoring transplanted BM-spheroids. IVM was performed using the same method described in A and obtained at DPT 0, 1, and 7. Mice were injected with Alexa Fluor 647–conjugated CD31 antibody (vessel staining shown in red) via tail vein before GFP BM-spheroid transplantation. Scale bars, 500 μm. Right panel shows high-magnification views of vessel-entrapped spheroids (upper) and GFP single cells (lower). Scale bar, 25 μm. Max, maximum; Min, minimum.
- Figure 6
MRI imaging of NHP BM-spheroids and immunohistochemical (IHC) staining of graft-bearing livers. A: In the NHP model, NHP BM-MNCs were labeled with ferucarbotran for 24 h and then cultured for 3 days to form BM-spheroids. Ferucarbotran-labeled NHP BM-spheroids were transplanted via the portal vein and observed by MRI at DPT 2. White elliptical regions indicate visible hypointense spots from ferucarbotran-labeled NHP BM-spheroids. B: Concurrent tissue ex vivo MRI and histological analysis. A monkey was sacrificed at DPT 17, and liver tissues were obtained for tissue ex vivo MRI and iron staining. Left panel: liver tissues sliced from the caudate lobe of the liver (arrows of top-right corner inset). Middle panel: ex vivo MRI imaging of the liver slices shown in the left panel. Right panel: Prussian blue staining for iron in the same tissue section; blue circles indicate the correspondence of hypointense spots and Prussian blue staining, and the upper and lower panels are magnified views of iron staining (blue). Scale bars, 100 μm. C: Livers were obtained from control-1, control-2, BM-spheroid-1, and BM-spheroid-2 at DPT 90, 90, 36, and 191, respectively. Live tissues were stained with anti-insulin, CD3, CD68, and CD31 antibodies to reveal NHP β-cells, T cells, macrophages, and vessels, respectively. Islets from normoglycemic BM-spheroid-1 and BM-spheroid-2 show intact morphology and are devoid of immune cell infiltration, whereas the control group of islets alone showed no insulin-positive β-cells or CD31-stained vessel cells and was occupied by immune cells. Scale bars, 100 μm. D: Quantification of the β-cell areas (%) in control-1, control-2, BM-spheroid-1, and BM-spheroid-2. H&E, hematoxylin-eosin.
- Figure 7
Blood glucose control by cotransplantation of islets and NHP BM-spheroids. A and B: Fasting blood glucose level and monkey C-peptide were measured in two monkeys that received intraportal transplants of allogeneic monkey islets (20,000 IEQ/kg) (A) and two monkeys that received cotransplants of allogeneic monkey islets (20,000 IEQ/kg) (B) and autologous recipient BM-spheroids via the portal vein. The control-1 and control-2 monkeys were insulin dependent until the end of the experiment (at DPT 93 and 98, respectively), whereas the BM-spheroid-1 monkey was insulin independent for 36 days (before sacrifice) and the BM-spheroid-2 monkey continued to maintain normoglycemia without insulin injection until the end of the experiment (at DPT 191). Blue line, blood glucose levels; red bar, monkey C-peptide; green bar, exogenous insulin. C: IVGTT. An IVGTT was performed at the indicated time points and examined before and after diabetes (DM) and 1, 2, and 6 months (mo) posttransplantation (post TPL). D: C-peptide secretion in response to glucose load. Before induction of diabetes, monkey C-peptide was normally detected after glucose injection; after induction of diabetes, monkeys showed C-peptide levels <1.0 ng/mL. E: The body weight change after islet transplantation in each recipient.
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