Molecular Modulation and Differential Characterization of Mammary Adipose Stem Cells towards Epithelialization
저자
발행사항
Chuncheon : Kangwon National University, 2014
학위논문사항
Thesis(Ph.D.)-- Kangwon National University : Department of animal Life Sciences 2014. 8
발행연도
2014
작성언어
영어
주제어
발행국(도시)
대한민국
형태사항
x, 112 L. : Illustrations ; 26 cm
일반주기명
강원대학교 논문은 저작권에 의해 보호받습니다
지도교수:나레시 쿠마 싱
References : L.94-111
소장기관
The research was designed to investigate the changes in gene expression pattern of mammary gland epithelial cells and adipose stromal cells with the changes in cellular microenvironment through epithelial induction mixture, adipogenic mixture and angiogenic mixture and also to justify the potential use of mammary adipose-derived stem cells for the regeneration of mammary glandular epithelial-like cells. Therefore, I conducted the whole studies under three different chapters. Chapter-I says about “Differentiation of adipose cells to epithelial lineage and epithelial cells to adipogenic lineage”. Chapter-I was composed of three different independent experiments, where the first experiment was conducted using mammary fat pad adipose derived stem cells isolated from Korean Black Goat mammary fat pad adipose tissues. The second and third experiment was conducted using 3T3L1 mouse adipose cells and MAC-T bovine mammary epithelial cells respectively to support our findings from the first experiment. The Chapter-II says about “Modulation of gene expression in mammary epithelial cells using angiogenic factors in presence or absence of mammary adipose stromal cells”. The chapter-II was composed of a detailed experiment to investigate the effect of angiogenic mixture for the differentiation of mammary epithelial cells in presence or absence of mammary adipose stromal cells. Finally, the Chapter-III says about “Transfection of K18 gene to CD44+ rat mammary adipose derived cells and subsequent infusion of the transfected cells to the rat aural space for epithelializaion”. Chapter-III basically investigated the transfection potential of mammary epithelial-specific gene(s) to mammary adipose-derived stem cells for the generation of mammary epithelial-like cells.
In Chapter-I, the Experiment-I was conducted to know and investigate the mechanism involved during mesenchymal to epithelial transition to unravel questions related to mammary gland development in prepubertal Korean black goat. We therefore, biopsied mammary fat pad and isolated adipose cells and characterized with stemness factors (CD34, CD13, CD44, CD106 and vimentin) immunologically and through their genetic expression. Furthermore, characterized cells were differentiated to adipogenic (thiazolidenediones and α-Linolenic acid) and epithelial (keratinocyte growth factor) lineages. Thiazolidenediones /or in combination with α-Linolenic acid demonstrated significant up-regulation of adipo-Q, PPAR-γ, CEBP-α, LPL, and resistin. Adipose stem cells in induction mixture (5 μg/ml Insulin, 1 μg/ml Hydrocortisone, 10 ng/ml epidermal growth factor) and subsequent treatment with 10 ng/ml keratinocyte growth factor revealed their trans-differentiating ability to epithelial lineage. From 2 d onwards, the cells under keratinocyte growth factor influenced cells to assume rectangular (2-4 d) to cuboidal (8-10 d) shapes. Ayoub-shklar stain developed brownish-red pigment in the transformed cells. Though, expressions of K8 and K18 were noted to be highly significant (p<0.01) but expressions of epithelial membrane antigens (EMA) and epithelial specific antigens (ESA) were also significant (p<0.05) compared to 0 d. Conclusively, epithelial transformations of mammary adipose stem cells would add up knowledge to develop therapeutic regimen to deal with mammary tissue injury and diseases.
Experiment-II was conducted for the further justification of the findings of Experiment-I. Aiming at mesenchymal to epithelial transition, we investigated the potential of keratinocyte growth factor (KGF) and bone morphogenetic protein (BMP)-6 separately for the induction of mesenchymal to epithelial transition in 3T3L1 mouse adipose cells and to trace the molecular events that probably upregulates during mesenchymal to epithelial transition induction. KGF successfully induced mesenchymal to epithelial transition through the up regulation of epithelial related genes and transcript expression on 3T3L1 cells. In contrast, BMP-6 plays completely reverse role through down regulation of all epithelial related genes and transcript expression. In KGF based treatment, seven genes (K8, K18, EpCAM, K5, K14, SMN1 and α-SMA) out of total eight genes were significantly (p<0.05/ p<0.01) up regulated. Immunostaining and immunoblotting also revealed significant (p<0.05/ p<0.01) expression of several epithelial-specific surface antigens and transcripts. Moreover, ayoub shaklar staining (specific to keratin) of KGF treated cells showed formation of keratin (reddish brown color) within cytoplasm of the cells whereas, the control and BMP-6 treated cells did not. Conclusively, KGF was observed to have the potential to enhance mesenchymal to epithelial transition and these clues could be used in the future research of cellular reprogramming and regenerative medicine
Experiment-III was performed to investigate the effect of adipogenic mixture for the differentiation and formation of cytoplasmic lipid droplets (CLD’s) in MAC-T mammary epithelial cells. CLD’s formation is a critical requirement for the onset of lactation and prevention of many diseases in the mammary gland. We therefore investigated the number of genes and transcripts that could be detrimental in upholding the formation of CLD’s in bovine mammary epithelial cells (MAC-T). We therefore, designed the present research to investigate the effect of alpha-linolenic acid (LA) plus Thiazolidenediones (TZD’s) in the formation of CLD’s in bovine mammary epithelial cells (MAC-T). MAC-T cells were proliferated in defined medium and upon confluence treated with differentiation medium containing LA (100 μM) and TZD’s (10 μM), and the medium was changed at every 48 h until 8 d. Control cells were cultured only in DMEM+2 % HS+1.1 % PS. Microscopically, it was quite apparent that LA plus TZD’s had successfully introduced CLD’s in cellular cytoplasm and that was further confirmed with Oil-red-O staining that showed significant (p<0.05) elution index signifying the amount of stain retained within. We also observed significant (p<0.05/P<0.01) up-regulation of adipogenic and down regulation of epithelial markers. In conclusion, 100 μM LA plus 10 μM TZD’s results formation of CLD’s and subsequent differentiation of MAC-T cells. In addition, evidence has been presented here that TZD’s and LA could absolutely alter and initiates a cellular spark in the mammary epithielial cells to undergo concerted with adipose program and develop shifts in the lipid metabolism to develop CLD’s efficiently and to increase the availability of fatty acids necessary for lipid synthesis by milk secreting cells.
In Chapter-II, the investigation was performed to find out the effect of angiogenic factors for the differentiation of MAC-T mammary epithelial cells in presence or absence of mammary adipose stromal cells under different culture models. Successful lactation mainly influenced by three major factors such as growth, proliferation and differentiation of mammary epithelia, proper functioning of stromal cells and abundant blood circulation. Thus, the balance in the presence of epithelial cells, adipose cells, endothelial cells, nerve cells and immune cells and their synergistic influence results a successful lactation. We believed that further understanding the influence of angiogenic factors in mammary parenchyma-stromal cells interaction would explore the hidden regulatory mechanism for the proliferation and differentiation of mammary epithelial cells. Therefore, we also designed our study to determine the effect of angiogenic factors VEGF plus T4 and/or Tetrac (angiogenic blocker) in MAC-T bovine mammary epithelial cells in three different culture conditions such as monolayer culture of MAC-T cells, trans-well culture (adipose at top and MAC-T at bottom) and mixed-culture (1:1 mixture of adipose and MAC-T cells). Both epithelial and adipose cells were seeded with a density of 2×106 cells per well in DMEM+10% FBS+1.1% PS and incubated for 24 h. Thereafter, the cells were exposed to angiogenic induction medium (DMEM+2%HS+1.1% PS+10 ng/mL bFGF+10 ng/mL EGF) for another 48 h. Finally, the cells were cultured again 48 h with four different treatment combinations (such as 50 ng/ml VEGF, 50 ng/ml VEGF+100 nmol/L T4, 50 ng/ml VEGF+10-7 M Tetrac and 50 ng/ml VEGF+100 nmol/L T4+10-7 M Tetrac) and one untreated control. The treated cells were harvested subsequently and were subjected to different analytical methods. In monolayer culture, only angiogenic markers and some epithelial markers showed significant up-regulation (P<0.05/P<0.01), and mammary epithelial-specific Keratin-18 (K18) got enhanced insignificantly with VEGF+T4 treatment and seemingly, 10-7 M tetrac is not enough to block the effect of angiogenic factors. In trans-well culture, only VEGF treated cells significantly (P<0.05/P<0.01) up-regulated few angiogenic transcript and down-regulated few epithelilal markers. VEGF+ T4 treated cells showed significant (P<0.05/P<0.01) up-regulation of most of the angiogenic and adipogenic markers and but down-regulated the mammary epithelial-specific marker K18. In this group also 10-7 M tetrac could not block the angiogenic effect but reduced the effect of angiogenic factors in different extent. In mixed-culture cells, most of the genes and markers lose their expression however, in control cells there was weak expression for different markers and epithelial marker K18 totally disappeared. However, VEGF+ T4 treated cells showed significant (P<0.05/P<0.01) up-regulation for few angiogenic and epithelial and in some cases adipogenic markers under different analytical methods. The cells treated in combination with blocker (Tetrac) caused inconsistent up/down-regulation of some transcript compared to the control cells, but it appears that the homogenous and direct haphazard mixture resulted the loss of characteristics features of mammary epithelial cells. Furthermore, the microarray gene expression profile was checked for the co-cultured mammary epithelial cells.
The microarray gene profiling showed, the VEGF alone mainly influenced the expression for adipogenic differentiation genes, lipid metabolism gene expression, and also cause reduction in the expression of cell adhesion molecules. VEGF+T4 treatment resulted up-regulation of endothelial cell adhesion molecules, cell proliferation molecules, and down-regulation of lipid metabolism molecules. Furthermore, VEGF alone up-regulated PPAR signal (specific for adipogenesis) and VEGF+T4 up-regulated WNT signal (known for endothelial growth and proliferation). Therefore, it could be concluded that VEGF alone might pose the role towards differentiation of the mammary epithelial cells and VEGF+T4 could influence the cells transformation towards endothelial-like cells. The angiogenic factors could not cause the loss of epithelial markers in absence of adipose cells. But, the systematic presence of adipose factors could influence the characteristics features of mammary epithelial cells and the haphazard and direct mixture of mammary epithelial cells with adipose stroma could cause the loss characteristics markers expression.
In Chapter-III, the research was conducted focusing on transfection of mammary epithelial-specific gene to mammary adipose stroma derived stem cells for the investigation of the potential uses of mammary adipose-derived stem cells for the regeneration of mammary epithelial cells. In this study, the mammary adipose tissue were surgically harvested from Slc:SD female rat of 10 week old (around 200 g live weight) and the adipose-derived cells were isolated using enzymatic digestion process with collagenase type-I and then comparatively smaller sized cells were collected through differential trypsinization process. After that, the mesenchymal stemness marker CD44+ cells were sorted using FACSAria-II. Finally, the CD44+ cells were transfected with mammary epithelial specific gene K18 and subsequently the transfection was confirmed by detecting GFP+ cells through fluorescence microscopy and thereafter, the downstream analysis of the K18 gene transfected cells was performed by puromycin antibiotic treatment, gene and protein expression profiling, Ayoub shklar staining and finally the cells were grafted to the rat aural space for in vivo study. In result, 25-30 % cells were found to be transfected when it was detected for GFP+ expression under fluorescence microscopy after 24 h of transfection. But, the gene and protein expression did not show significant difference between control and transfected cells. However, the ayoubl shklar staining showed reddish brown color formation in some cells, but all of the cells had shrinkage cytoplasm (spider shape) and seemed to get detached chronologically from the surface of the culture dish. Therefore, it could be concluded that the conversion of mammary adipose CD44+ cells towards epithelial lineage need to be standardized further for having sound transfection rate and subsequent survivability of the cells.
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