3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

Abstract

Bone marrow cells (BMC) have been shown to migrate into injured sites for parenchymal repair. However, the extent of BMC involvement is controversial. To determine whether and to what extent BMC contribute to renal parenchymal repair, we employed three-dimensional (3D) fluorescent confocal microscopy/video in renal warm and cold ischemia/reperfusion (I/R) injury using enhanced green fluorescent protein transgenic rats and their radiation chimeras. After induction of renal warm I/R injury in chimeras, BM-derived renal tubular cells were found in 2D microscopy as isolated single cells or clusters of 2-3 cells. Likewise, cold I/R injury resulted in hostderived tubular cells with frequencies B0.2%. However, stringent confocal microscopic analysis and 3D image construction revealed that BM-derived tubules identified in 2D images were frequently artifacts of overlapping cells separately stained with different markers. The actual frequency in 3D analysis was approximately onefourth of that seen in 2D analysis. 3D confocal imaging precisely detected BM-derived tubular epithelial cells and could be useful to study BMC contribution to tissue repair. Although there are abundant data on the phenotype, isolation, culturing, expansion, and differentiation potential of stem cells in the bone marrow, their role and functional relevance in normal adult life or regenerative parenchymal cell turnover is unclear. In addition, many of the findings in this new field are controversial, partly due to the inherent difficulties of techniques used to assess the plasticity of these cells.In this study, transgenic rats carrying the enhanced green fluorescent protein (EGFP) transgene were used to demonstrate three-dimensional (3D) structure analysis of the differentiation of BMC to renal tubular epithelial cells after renal ischemia/ reperfusion (I/R) injury. The results suggest that BMderived cells can differentiate into renal tubular epithelial cells after I/R injury; however, the frequency is considerably less with 3D analysis than that identified with conventional 2D microscopic analyses of thin sections. Materials and methods AnimalsEGFP-transgenic and WT Sprague-Dawley (SD) rats, originally generated by Dr Masaru Okabe (University

Inside Lab Invest

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Characterization of Transplanted Green Fluorescent Protein+ Bone Marrow Cells into Adipose Tissue

“…The successful creation of a GFP chimera was confirmed by detecting GFP ϩ peripheral blood mononuclear cells (PBMCs) with flow cytometry in the blood. The percentage of GFP ϩ PBMCs in WT recipients quickly increased and reached Ͼ95% by 100 days [21]. At 116 -402 days after transplantation, subcutaneous and intra-abdominal fat tissues were obtained from 26 GFP radiation chimeras and used in this study.…”

Simultaneous bone marrow and intestine transplantation promotes marrow-derived hematopoietic stem cell engraftment and chimerism

“…Green fluorescent protein (GFP)-transgenic and wild-type (WT) Sprague Dawley (SD) rats originally generated by Dr Masaru Okabe (University of Osaka, Japan) [18][19][20] were obtained from Japan SLC (Hamamatsu, Japan). The expression of GFP was under the control of the cytomegalovirus enhancer and the chicken ␤-actin promoter derived from an expression vector, pCAGGS.…”

“…The expression of GFP was under the control of the cytomegalovirus enhancer and the chicken ␤-actin promoter derived from an expression vector, pCAGGS. [18][19][20] Inbred BN (RT1 n ), LEW (RT1 l ), and ACI (RT1 a ) rats weighing 180 to 250 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). All animals were maintained in a laminar flow animal facility at the University of Pittsburgh and fed with a standard diet ad libitum.…”