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First published online 17 February 2004
doi: 10.1242/jcs.00926

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Epidermal cells accelerate the restoration of the blood flow in diabetic ischemic limbs

Chunhua Jiao^1,*, Sarah Bronner^2,*, Keri L. N. Mercer^2,*, Don D. Sheriff¹, Gina C. Schatteman¹ and Martine Dunnwald^2,

¹ Department of Exercise Science, The University of Iowa, Iowa City, IA 52242, USA
² Joe Marshall Laboratory, Department of Dermatology, The University of Iowa, Iowa City, IA 52242, USA

View larger version (37K): [in a new window]   Fig. 1. FACS analysis of mouse epidermal cells sorted into progenitor and TA cells. The figure shows gates for EpPCs and TA cells as described in Materials and Methods.

View larger version (136K): [in a new window]   Fig. 2. Incorporation of freshly isolated epidermal cells into the vasculature. Freshly isolated epidermal progenitor cells (A,B) and transient amplifying cells (C) were labeled with CM-DiI (red fluorescent dye) and injected into a mouse ischemic hindlimb. Two weeks later, biopsies of skin (A,D) and ischemic muscles (B,C,E) were immunostained for CD31 (to identify endothelial cells; green) and keratin 14 (blue, a marker for epidermal basal cells; A, arrowheads). CM-DiI-labeled (red) cells, co-labeled CM-DiI and anti-CD31 (yellow) cells, but not keratin-14-positive (blue) cells can be seen in the vessels. Arrows indicate two capillaries enlarged in (D). No injected cells (red) could be seen in the vasculature (green) in the contralateral control muscle (E). Notice that the two red dots in (A) correspond to autofluorescent hair shaft cut in oblique sections and are not CM-DiI-injected cells. Scale bars, 25 µm.

View larger version (98K): [in a new window]   Fig. 3. Confocal images of epidermal cells incorporating into the vasculature. Epidermal cells were isolated, labeled with the red fluorescent dye CM-DiI and injected into diabetic mice with an ischemic leg. One month after the injection, mice were perfused with FITC-tagged BSLB4 (a labelled lectin specific for mouse endothelial cells) before tissue was harvested. Pictures were taken with a confocal microscope. For this particular example, a z series of 60 focal plans was acquired 1.5 µm apart. (a-f) Consecutive stacks of six images each. Notice the red fluorescent cells (epidermal cells labeled with CM-DiI, arrows) incorporated into the vasculature delineated by the green fluorescence. Scale bars, 25 µm.

View larger version (160K): [in a new window]   Fig. 4. Incorporation of cultured epidermal cells into the vasculature. Cultured epidermal progenitor cells (B) and transient amplifying cells (A) were labeled with CM-DiI (red fluorescent dye) and injected into a mouse ischemic hindlimb. Five weeks later, biopsies of ischemic muscles were immunostained for CD31 (to identify endothelial cells; green). CM-DiI-labeled (red) cells, co-labeled CM-DiI and anti-CD31 (yellow) cells can be seen in the vessels. No injected cells (red) could be seen in the vasculature (green) in the contralateral control muscle (C). Scale bars, 25 µm.

View larger version (74K): [in a new window]   Fig. 5. Laser Doppler blood-flow images. Representative images of control (buffer or fibroblast injected) and diabetic animals injected with EpPCs. Mice were monitored before the surgery to verify the integrity of the blood flow and every other day after the surgery to evaluate the restoration of the blood flow. Dark blue areas represent area with no flow and white areas represent regions with the highest flow. Notice the lack of flow immediately after the surgery. After 12 days, the flux was restored to 33% in the control buffer-injected animals, compared with 55% in the progenitor-cell-injected animals.

View larger version (18K): [in a new window]   Fig. 6. Restoration of the blood flow in surgically induced ischemia in diabetic mouse hindlimbs. The data are expressed as the percentage of blood flow in the operated limb relative to the contralateral unoperated limb from the same animal. Fluxes were measured using a scanning laser Doppler and means of between three and five readings were calculated for each animal at each time point. Averages of means for 4-16 animals were plotted as a function of time. Error bars represent standard error. *, Unsorted cells significantly different to fibroblast-injected and control animals by one-way Anova (P<0.05); , unsorted and progenitor cells significantly different to fibroblast-injected and control animals by one-way Anova (P<0.05); ¥, unsorted cells, progenitor cells and TA cells significantly different from fibroblast-injected and control animals by one-way Anova (P<0.05). The linear regression of the flow from progenitor-cell-injected animals was also significantly different from the control animals by one-way Anova (P<0.05).

View larger version (190K): [in a new window]   Fig. 7. Histology and vascularization of muscle in ischemic hindlimbs 14 days after iliac artery ligation. Hematoxylin and eosin stained 7 µm transverse sections of muscle in the lower hindlimb at the level of the distal gastrocnemius muscle. Sections were incubated with BSLB4 and reacted with Vector Red to visualize blood vessels (bright red). (A-C) A limb treated with progenitor cells. (D) A fibroblast-treated limb. (A) Healthy muscle. (B) Recovering muscle. (C) Severely injured recovering muscle with inflammatory infiltrate. (D) Necrotic muscle. Scale bar, 50 µm.

View larger version (17K): [in a new window]   Fig. 8. Restoration of the blood flow in surgically induced ischemia in non-diabetic mouse hindlimbs. The data are expressed as the percentage of blood flow in the operated limb relative to the contralateral unoperated hindlimb from the same animal. Fluxes were measured using a scanning laser Doppler and means of between three and five readings were calculated for each animal at each time point. Averages of means for 5-11 animals were plotted as a function of time. Error bars represent standard error. *, Statistically significant difference from the control animals by one-way Anova (P<0.05) at days 2-6 for the TA group, and at days 8-10 for the progenitor group.

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