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Adipose derived osteoblasts cells from fat tissues
 

Adipose derived osteoblasts cells from fat tissues

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    Adipose derived osteoblasts cells from fat tissues Adipose derived osteoblasts cells from fat tissues Presentation Transcript

    • SURFACE PROTEIN CHARACTERIZATION OF OSTEOBLASTS AND ADIPOSE DERIVED OSTEOBLASTS CELLS Vij Senthilnathan
    • OBJECTIVE
      • A comparative analysis of primary osteoblasts from the iliac crest and osteogenic differentiated mesenchymal stem cells from adipose tissue, using osteoblast-specific protein expression.
    • BONE MARROW STEM CELLS – NO!!! NO!!!
      • Two types of stem cells for tissue engineering :
        • Embryonic stem cells - Problems of cell regulation & ethics
        • Autologous stem cells - Immunocompatible & No ethical issues
      • Bone Marrow Stem Cells
        • Autologous stem cells from bone marrow : Proven experimentally promising
        • Bone marrow stroma : Heterogeneous in composition
          • Hematopoietic stem cells
          • Mesenchymal stroma
        • But traditional bone marrow procurement :
          • Painful
          • Requires general / spinal anesthesia
          • Yields low MSCs upon processing
            • App.1 MSCs per 100,000 adherent stromal cells
        • Ex-vivo expansion means
          • Expensive & Time consuming
          • Cross contamination & loss
    • AN IDEAL SOURCE OF AUTOLOGOUS STEM CELLS
      • Abundant quantities
      • Minimal invasive procedure (less patient discomfort)
      • High cell number
      • Differentiate into multiple lineage
      • Safe/ Effective to transport
      • Fit the GMP guidelines
      • No ethical problems
      • Adipose tissue may represent such a source
      • Like bone marrow,
        • Derived from embryonic mesoderm
        • Heterogeneous stromal cell population
      Note: App. 400,000 liposuction in US/yr
    • BONE BIOLOGY Osteoblasts (Support cells) Osteoclasts (Remodeling cells)
      • Arise from osteoprogenitor cells
      • Mitotic division
      • Bone matrix synthesis & mineralization
      • Derived from hematopoietic lineage
      • Bone resorption
      • These are osteoblasts incorporated in the newly formed osteoid
      • Transduce messages to the osteoblastic cells
      Secretion ALP BONE CELLS Osteocytes (Support cells) MSCs BMP Differentiation Osteoprogenitor cells Activation by growth factors Cbfa1 Active osteoblast Bone mineralization facilitators: Osteocalcin, Collagen type 1 & Osteopontin
    • PROJECT OUTLINE Study the gene & protein expression profile ILIAC CREST 1.TOTAL KNEE REPLACEMENT SURGERY 2.TOTAL HIP REPLACEMENT SURGERY Osteoblasts like cells Adipose tissue Osteogenic conditions Bone samples from iliac crest Osteoblasts cells Bone media
    • PATIENTS DETAILS ADSCs: Adipose derived stem cells BMSCs: Bone marrow stem cells OB: Osteoblasts ADOBs: Adipose derived Osteoblasts cells Viability test n= 6 donors 81% 72% 28% OBs 81% 68% 30% BMSCs 84% 70% 28% ADSCs RL (30min) Saline (30min) PBS (30min) CELL TYPE
    • ISOLATION & CULTURE OF ADIPOSE TISSUES
      • Human subcutaneous adipose tissue:Transported in anticoagulant or EDTA within 2h post-surgery. Minced & digested in digestion buffer:1-2 hrs at 37’c.
      • Stromal vascular fraction incubated in Osteogenic media: αMEM, supplemented with 10% FBS, 10mM ß-glycerophosphate, 0.1 uM dexamethasone, 1% antibiotics/antimycotic solutions, 50 uM ascorbate-2-phosphate (Zuk et al 2001)
      Fat tissue Removal of vessels and fascia Collagenase digestion
    • ISOLATION & CULTURE OF OSTEOBLASTS
      • Bone samples : Transported in
      • anticoagulant or EDTA within 2h post-surgery
      • Extensive 1xPBS washing – Remove hematopoietic contaminants
      • Collagenase digestion: 1-3 hrs at 37’c
      • Neutralization with media and centrifugation
      • Bone pieces plated in T-25 flasks containing in αMEM, 10% FBS, 250ul Vitamin C,1% antibiotic / antimycotic solutions
      • Non-adherent cells were removed after 12-24 hrs
    • MORPHOLOGY OF THE CELLS
      • Representative images of human OB & ADOB cells at P4. The cells at each passage were examined under light microscopy
      • Figure a: 100% confluent osteoblasts (OB) at day 10;
      • Figure b: 100% confluent adipose derived osteoblasts cells (ADOB) at day 7
      a. OB 10X LM b. ADOB 10X LM
    • ADIPOSE DERIVED OSTEOBLAST CELLS
      • The proliferation of adipose tissue in response to the osteogenic media. Data expressed as mean +/- SEM (n=5). Statistical analysis was performed using a repeated measure ANOVA followed by Newman – Keuls. (Prism 4.0C for Word, Graphpad, CA, USA)
      • *** P value <0.05.
      ***
    • OSTEOBLASTS
      • The proliferation of primary osteoblasts. Data expressed as mean +/- SEM (n=5). Statistical analysis was performed using a repeated measure ANOVA followed by Newman – Keuls. (Prism 4.0C for Word, Graphpad, CA, USA)
      • *** P value <0.05
      ***
    • HEMATOXYLIN & EOSIN STAINING
      • 150,000 cells/PD of cells were grown in petridishes overnight
      • Extensive washing with 1xPBS
      • Fixed with 2% PFA
      • Stain in Harris Hematoxylin solution
      • Differentiate in 1% acid alcohol solution
      • Treat in 0.2% ammonia water solution
      • Counter stain in eosin solution
      • Dry O/N & view under light microscope
      Representative images of human OB & ADOB cells at P3. Representative of n=3 donors.Blue purple stains the nucleus & bright pink stains the cytoplasm
    • IMMUNOFLUORESCENCE Osteocalcin & Nucleostemin Histochemical detection of protein expression by human OB & ADOB cells at P4. They were fixed and immunohistochemically stained with Osteocalcin & Nucleostemin. Representative of n=5 donors
      • Cell were grown in cover slips
      • Fixed with 2% PFA & wash
      • Permeabilize with 0.2% Triton X 100 treatment
      • 2 hrs blocking – 1% BSA in 1x PBS
      • Primary antibody: Osteocalcin & Nucleostemin (1:50 dilution) – O/N (4’c)
      • 5 minute 1xPBS-T washing - Thrice
      • FITC-conjugated secondary antibody 1:100 dilution – 2 hrs at RT (dark)
      • Washing and mounting with glycerol medium
      • Antibody source: Santa Cruz Biotechnology,Inc. Switzerland
    • REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION
      • (i). 1ug/ul RNA was used. All the analysis was done using Qiagen kit.
        • RNA isolation kit
        • Omniscript TM Reverse Transcriptase kit
        • HotStar Taq ® Master Mix PCR Kit.
      • (ii). All the primer sequences were determined using established gene bank sequences. Primers were obtained from Microsynth, Switzerland.
      • Characterization of OBs & ADOBs :
      • Osteoblast markers:
        • GAPDH (endogenous control) 702 bp,Osteocalcin (OC) – 297 bp,Core Binding Factor1 (CBFA1 or HUNX2) – 256 bp,Alkaline phosphatase (ALP) – 356 bp, Collagen type I- (599bp)
        • Stem cell markers:
        • Nucleostemin (98bp), CD 10 (459bp), CD13 (449bp), CD44 (674bp), CD59 (439bp), CD105(377bp) and CD166(157bp)
    • GENE EXPRESSION BY HUMAN OB FOR OSTEOCALCIN, ALP, CBFA1, GAPDH & COLLAGEN TYPE 1 n=5 ADOB & OB : Collagen type 1
    • GENE EXPRESSION BY HUMAN ADOB FOR OSTEOCALCIN, ALP, CBFA1, GAPDH n=5 ADOB: Osteocalcin & GAPDH ADOB: 1-7; OB: 8,9; Endothelial cells: 10 Osteocalcin & CBFA1 ADOB: ALP & Osteocalcin
      • GENE EXPRESSION BY HUMAN ADOB FOR CD10, CD59, CD44, CD105,CD13,CD166,GAPDH
      • n=5
      OB : CD13 & GAPDH ADOB: CD10 & CD59 ADOB: CD44 & CD105 ADOB: CD13 & CD166
    • GENE EXPRESSION BY HUMAN OB & ADOB FOR NUCLEOSTEMIN n=5 OB ADOB
    • WESTERN IMMUNOBLOTTING
      • Protein isolation : 50ul RIPA (Rapid Immunoprecipitation Assay) buffer
        • 50 mM TrisHCl pH7.4,
        • 150 mM NaCl
        • 2 mM EDTA
        • 1% NP-40 (Nonidet P-40)
        • 0.1% SDS
      • Centrifugation at 1200rpm for 15minutes at 4’c and supernatant containing proteins were stored at –20’c until use
      • Protein concentration determined by Lowry’s method using BSA as standard
      • 25ug proteins were loaded to each well and separated using NuPAGE 4-12% Bis-Tris gel and transferred to nitrocellulose paper
      • Blocking with skimmed milk solution
      • Washing and O/N primary (Nucleostemin, Osteocalcin and GAPDH) at 4’c
      • Peroxidase conjugated secondary antibody for 2 hrs at RT
      • Blots visualized using ECL Chemiluminescence kit and auto radiographic exposure
      • Immunoblot detection of proteins expressed by human OB & ADOB cells. Total cell lysates from donors were electrophoresed on 4-12% Bis-Tris polyacrylamide gels, transferred and immunoblotted with antibodies directed against following antigens: Nucleostemin ( 62kDa) & GAPDH (37kDa). Representative of n= 4.
      • Antibody source: Santa Cruz Biotechnology,Inc. Switzerland
      WESTERN BLOTTING Nucleostemin ADOB 1-4 OB 5-8 Nucleostemin ADOB 1-3 OB 4-6
    • DISCUSSION
      • In this study, we defined the gene and protein expression profile of human osteoblasts cells and adipose derived osteoblasts cells in vitro . The expression pattern of various primers and antibodies were studied in both the cell types by Immunohistochemistry, RT-PCR and Western blotting.
      • Type I collagen and ALP are early markers of osteoblasts and the expression of these proteins by OB & ADOB cells have confirmed the osteogenic differentiation in our study.
      • Osteocalcin has been identified as the only osteoblasts specific gene which regulates the function of fully differentiated osteoblasts. Osteocalcin gene is regulated by its transcription factor called Cbfa1, belonging to the Runt family . Cbfa1 and Osteocalcin are the best suited osteogenic markers. Cbfa1 is strongly expressed by both the cell types but the expression of Osteocalcin is significantly less in ADOB in comparison to OB cells.
      • RT-PCR analysis revealed that the non-differentiated ADSCs contained different types of stromal cells with a large variety of CD marker expression.The pattern of expression of ADOB for all of the listed stem cell markers except CD13 were significantly positive.
    • DISCUSSION
      • Nucleostemin is a p53 binding protein which plays a significant role in proliferation of stem cells and cancer cells. Although nucleostemin has been associated for its cell proliferation regulation in adult stem cells, embryonic stem cells and cancer cells, we believe that our data supports a role other than in proliferation. Previous reports has demonstrated that nucleostemin expression was persistent over a long period of time in human BMSCs culture.
      • The expression of nucleostemin in OB and ADOB may be due to the fact that the cells are undergoing proliferation and have not been fully differentiated. But recent work also shows that cells proliferate after nucleostemin expression is lost, which further questions the role of nucleostemin in proliferation. This suggest that nucleostemin plays a role other than proliferation in differentiated cells and that may be the possible reason for its expression in OB and ADOB cells in our study.
      • Our results are a striking contrast to other studies and thus begins a new debate for the possible reason for its expression in cells other than adult stem cells and cancer cells.
    • CONCLUSION
      • We believe that the adipose tissue may represent as an another source for multipotent cells and will be a promising approach for future tissue engineering strategies and cell based therapies, due to its general availability and low donor morbidity. The current findings now identify the adipose tissue as another site containing stromal cells.However, future studies are necessary to establish the role of permanent differentiation of these multilineage potential cells.
    • Next phase …
      • The honeycomb collagen was found to be an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy. The differentiation of osteoblasts like cells from adipose tissue in honey-comb collagen scaffolds will be next step of this project.
    • SEMINARS & JOURNALS
      • Results accepted at 10th European Society of Trauma and Emergency Surgery (ESTES) 2009, Antalya, Turkey
      • Results accepted at NJ stem cell symposium 2009, New Jersey, USA
      • Results presented at the National Symposium of Trauma and Emergency Surgery, MIOT Hospitals, India
      • Paper submitted for European Journal of Trauma and Emergency Surgery
    • THANK YOU