Angioblast Derived from ES Cells Construct Blood Vessels and Ameliorate Diabetic Polyneuropathy in Mice

Abstract

Background: Although numerous reports addressing pathological involvements of diabetic polyneuropathy have been conducted, a universally effective treatment of diabetic polyneuropathy has not yet been established. Recently, regenerative medicine studies in diabetic polyneuropathy using somatic stem/progenitor cell have been reported. However, the effectiveness of these cell transplantations was restricted because of their functional and numerical impairment in diabetic objects. Here, we investigated the efficacy of treatment for diabetic polyneuropathy using angioblast-like cells derived from mouse embryonic stem cells.

Methods and results: Angioblast-like cells were obtained from mouse embryonic stem cells and transplantation of these cells improved several physiological impairments in diabetic polyneuropathy: hypoalgesia, delayed nerve conduction velocities, and reduced blood flow in sciatic nerve and plantar skin. Furthermore, pathologically, the capillary number to muscle fiber ratios were increased in skeletal muscles of transplanted hindlimbs, and intraepidermal nerve fiber densities were ameliorated in transplanted plantar skin. Transplanted cells maintained their viabilities and differentiated to endothelial cells and smooth muscle cells around the injection sites. Moreover, several transplanted cells constructed chimeric blood vessels with recipient cells.

Conclusions: These results suggest that transplantation of angioblast like cells induced from embryonic stem cells appears to be a novel therapeutic strategy for diabetic polyneuropathy.

Figure 1

Differentiation and purification of angioblast-like cells from mouse ES cells. (a) Spherical colonies of ES cells (left) were flatly outspread (right) after 4 days of culture on gelatin. Bar: 30 μm. (b) Fluorescence-activated cell sorting analyses showed that a population of cells expressing Flk1 was estimated at 10–20% (left) and came up to 55–63% by magnet associated cell separation (right). (c) Reverse-transcription PCR analyses showed that Flk1 mRNA expression in population selected as Flk1 positive cell was almost 30 times higher than that in population selected as Flk1 negative cell (P < 0.001). (d) Reverse-transcribed PCR analyses showed both mesoderm and angioblast markers, that is, Brachyury (T), Flk1, Flt1, Tie1, Tie2, VE-cad, PU.1, SCL/Tal1, and Lmo2, which were expressed in Flk1 positive cells. On the other hand, PECAM, which is considered as a marker of relatively differentiated endothelial cell, was not detected in Flk1 positive cells after the 4 days of differentiation.

Figure 2

Transcript levels of angiogenic and neurotrophic factors in angioblast-like cells induced from ES cells (ES-ABs). Sorted ES-ABs expressed angiogenic and neurotrophic factors: VEGF-A, PDGF-A, FGF2, NGF, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), Neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF). Each expression level of the factors compared Flk1 positive cells with Flk1 negative cells, PA6 cells, and IMS32 cells. PA6: a cell line of mouse mesenchymal stem cell and IMS32: a cell line of immortalized mouse Schwann cell. Flk1⁺: Flk1 positive cells and Flk1⁻: Flk1 negative cells. ^∗ P < 0.05 versus Flk1⁻ cells, ^## P < 0.005 versus PA6 cells, and ^†† P < 0.005 versus IMS32 cells.

Figure 3

In vitro differentiation potential of angioblast-like cells induced from ES cells (ES-ABs). After a 4-day culture on gelatin, ES-ABs formed many colonies. Immunocytochemistry, a smooth muscle cell marker, α-SMA, was detected at some colonies (a), and an endothelial marker, PECAM, was at the other colonies (b). Red: α-SMA and green: PECAM. Bar: 100 μm.

Figure 4

Tube forming assay of angioblast-like cells induced from ES cells (ES-ABs) on Matrigel. To elucidate whether ES-ABs themselves are enabled to form blood vessels, sorted ES-ABs and Flk1 negative cells were, respectively, seeded on Matrigel. Two days later, cells were visualized with isolectin IB4, a stain of endothelial cell. (a) Although endothelial cells stained with IB4 were rarely induced from Flk1 negative cells (right), heavy numbers of endothelial cells were induced from ES-ABs and deployed cobblestone-like pattern (left). Bar: 100 μm. Red: isolectin IB4. (b) In ES-AB culture, a part of cells stained with IB4 structured vessel like tube formation (left 2 images). On the other hand, cells forming a line were not stained with IB4 in culture of Flk1 negative cells (right 2 images). Bar: 100 μm. Red: isolectin IB4 and blue: DAPI.

Figure 5

The transplanted angioblast-like cells induced from ES cells (ES-ABs) constructed blood vessel walls and capillaries. Immunohistochemical staining revealed that transplanted GFP-expressing ES-ABs were still engrafted 4 weeks after transplantation. (a) The engrafted cells distributed in vessel wall between hindlimb muscle fibers and coexpressed α-SMA. Green: GFP and red: α-SMA. M: muscle and V: lumen of a blood vessel. Bar: 20 μm. (b) Some of GFP positive cells resided in gaps between soleus muscle fibers and coexpressed PECAM. Green: GFP and red: PECAM. White arrows: GFP negative capillaries and yellow arrowheads: GFP positive capillaries. Bar: 20 μm.

Figure 6

Capillary number to muscle fiber ratios in soleus muscles. (a) The vasculatures were visualized by Alexa594-conjugated isolectin IB4 in soleus muscles. More capillaries existed in soleus muscle of transplanted diabetic mice (right) compared to those of nontransplanted control diabetic mice (left). Red: isolectin IB4. Bar: 50 μm. DM-saline: saline-injected limb in diabetic mice and DM-AB: limbs transplanted angioblast-like cell derived from ES cells in diabetic mice. (b) Quantitative analyses revealed that the capillary number to muscle fiber ratios in the saline-injected sides of diabetic mice were significantly reduced compared with those of normal mice (N). Transplantation of angioblast-like cell derived from ES cells (ES-ABs) significantly augmented the ratios in transplanted limbs compared with those in saline-injected side limbs in diabetic mice (DM). Transplantation of ES-ABs into normal mice showed no significant differences. S: saline treated limbs, AB+: limbs transplanted ES-ABs, and AB−: contralateral limbs transplanted ES-ABs. Results are means ± SD. ^∗ P < 0.05 versus S in N (N-S) and ^† P < 0.05 versus S in DM (DM-S). N = 4 in N-S and n = 3 in DM-S (P = 0.0004). N = 3 of AB+ in DM and n = 5 of AB− in DM (P = 0.0276 represents DM-S versus AB+ in DM). N = 4 of AB+ in N and n = 4 of AB− in N (P = 0.9609 represents N-S versus AB+ in N).

Figure 7

Blood flow of sciatic nerves and plantar skins were ameliorated by transplantation of angioblast-like cell derived from ES cells (ES-ABs). At a time point of 12 weeks of diabetes (before treatment), blood flows of plantar skins (a) and sciatic nerves (b) in diabetic mice significantly decreased compared with those in normal mice, and, 4 weeks after transplantation (after treatment), the decreases were ameliorated in transplanted limbs of diabetic mice. However, administration of ES-ABs did not alter blood flow in those of normal mice. N: normal mice limbs, DM: diabetic mice limbs, S: saline-injected limbs, AB+: limbs transplanted ES-ABs, and AB−: contralateral limbs transplanted ES-ABs. Results are means ± SD. ^# P < 0.05 versus pretreatment N. ^∗ P < 0.05 versus posttreatment S-treated N, ^† P < 0.05 versus posttreatment S-treated DM. n = 4–6 in each pretreatment group and n = 7–10 in each posttreatment group.

Figure 8

Impaired sensory perceptions in diabetic mice were ameliorated by angioblast-like cells induced from ES cells (ES-ABs) transplantation. Stimuli with frequencies of 5 Hz (a), 250 Hz (b), and 2000 Hz (c) evoked excitations of C-fiber, Aδ-fiber, and Aβ-fiber, respectively. Before cell transplantation, CPTs with all kinds of stimuli in diabetic mice had significantly increased compared with those in normal mice, representing hypoalgesia in diabetic mice. Four weeks after the transplantation of ES-ABs, these deficits in sensation were significantly improved in diabetic mice compared with saline-treated diabetic controls. The transplantation of ES-ABs into normal mice did not induce significant changes in CPTs. N: normal mice limbs, DM: diabetic mice limbs, S: saline-injected limbs, AB+: limbs transplanted ES-ABs, and AB−: contralateral limbs transplanted ES-ABs. Results are means ± SD. ^# P < 0.05 versus pretreatment N, ^∗ P < 0.05 versus posttreatment S-treated N, and ^† P < 0.05 versus posttreatment S-treated DM. n = 8–10 in each nondiabetic group and n = 7–9 in each diabetic group.

Figure 9

Transplantation of angioblast-like cells induced from ES cells (ES-ABs) improved delayed nerve conduction velocities (NCVs) in diabetic mice. Motor nerve conduction velocity (MNCV) (a) and sensory nerve conduction velocity (SNCV) (b) of diabetic mice were significantly delayed compared with those of normal mice after 12 weeks duration of diabetes. The delays in MNCV and SNCV were significantly restored by ES-ABs transplantation. However, administrations of ES-ABs did not alter NCVs in normal mice. N: normal mice limbs, DM: diabetic mice limbs, S: saline-injected limbs, AB+: limbs transplanted ES-ABs, and AB−: contralateral limbs transplanted ES-ABs. Results are means ± SD. ^# P < 0.05 versus pretreatment N, ^∗ P < 0.05 versus posttreatment S-treated N, and ^† P < 0.05 versus posttreatment S-treated DM. n = 6 in each pretreatment group and n = 7–10 in each posttreatment group.

Figure 10

Intraepidermal nerve fibers (IENFs) were preserved by angioblast-like cells induced from ES cells (ES-ABs). (a) IENFs in plantar skins were visualized with PGP9.5 antibody (red). After 15 weeks of diabetes, IENF densities were decreased in diabetic mice (upper) and the impairment was restored by ES-ABs transplantation (lower). DM-saline: plantar skin of saline-injected limbs in diabetic mice and DM-ES-AB: plantar skin of limbs transplanted ES-ABs in diabetic mice. White arrowheads: IENFs. (b) Quantification of IENF densities demonstrated that IENFs were significantly decreased in diabetic mice compared with those in normal mice at 12 weeks of diabetes and this decrease was significantly ameliorated by transplantation of ES-ABs. However, administration of ES-ABs did not change IENF densities in normal mice. N: normal mice limbs, DM: diabetic mice limbs, S: saline-injected limbs, AB+: limbs transplanted ES-ABs, and AB−: contralateral limbs transplanted ES-Abs. Results are means ± SD. ^## P < 0.005 versus pretreatment N, ^∗∗ P < 0.005 versus posttreatment S-treated N, and ^† P < 0.05 versus posttreatment S-treated DM. n = 3-4 in each group.