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  • 1. Human Inferior Turbinate : An Alternative Tissue Source of Multipotent Mesenchymal Stromal Cells Presenter –Madan Gupta Guide –Prof S C Sharma
  • 2. INTRODUCTION  MSCs (mesenchymal stromal cells) - plastic adherent fibroblast-like cells with extensive proliferative capacity and differentiation potential.  Types of stem cells: Embryonic  non-embryonic. Induced pluripotent stem cell (iPSC)
  • 3. International Society of Cellular Therapy  Three criteria: 1)MSCs must be adherent to plastic under standard tissue culture conditions. (2)MSCs must express certain cell surface markers such as CD73, CD90, and CD105, and lack expression of other markers including CD45, CD34, CD14, or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. (3)MSCs must have the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts under in vitro conditions.
  • 4. Biological characteristics of MSCs  The ability to home to sites of inflammation following tissue injury when injected intravenously  The ability to differentiate into various cell types  The ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation  The lack of immunogenicity and the ability to perform immunomodulatory functions
  • 5. MSC roles in vivo
  • 6. Immunoregulatory properties in vitro
  • 7. Therapeutic potential
  • 8. Clinical applications of MSCs  Le Blanc K et al- haploidentical MSCs- aGVHD of the gut and liver.  Ringdén O et al-6 gut,1 skin, 1 liver
  • 9.  The best-characterized MSC population - originating from bone marrow.  Alternative sources.  preliminary studies- human turbinate fibroblasts - growth in a chondrogenic medium acquire a chondrogenic phenotype
  • 10. HYPOTHESIS  In a previous investigation- fibroblasts from inferior turbinate tissue were plastic-adherent and differentiated into chondroid cells when grown in a chondrogenic medium- expressing the chondrogenic markers aggrecan and type II collagen as assessed by RT-PCR  Fibroblasts isolated from the inferior turbinate are multipotent MSCs- testing their capacity to differentiate into chondrogenic, osteogenic, adipogenic, and neurogenic cells.
  • 11. Materials and Methods Cell Isolation  0.0366 g- inferior turbinate tissues- discarded tissue - 10 patients who underwent septoplasty and partial turbinectomy.
  • 12. Washing of tissue & culturte  three times with antibiotic-antimycotic solution.  twice with phosphate buffered saline (PBS) solution.  75-mm2 cell culture disc(20mL of DMEM (Dulbecco’s Modified Eagle Media, Gibco& 10 percent FBS was added)  incubated in a 37°C, 5 percent CO2 incubator for 3 weeks,  The fibroblasts attached to the bottom of the culture dishes were obtained. test for chracterization of MSCs
  • 13. Multilineage Differentiation Potential of hTMSCs  Chondrogenesis  seeding cells onto polycaprolactone sponges.  Chondrogenic supplements TGF-b1 IGF-1  DMEM supplemented with 10% fetal bovine serum (FBS)  100 nM dexamethasone  50 mM ascorbic acid-2 phosphate  50 mg/mL insulin transferrin sodium selenite  1 mM sodium pyruvate  40 mM proline  2 mM Lglutamine
  • 14. osteogenic differentiation  low-glucose DMEM supplemented with 10% FBS.  100 U/mL penicillin.  100 mg/mL streptomycin.  0.1 mM dexamethasone.  50 mM ascorbate-2 phosphate  10 mM b-glycerophosphate
  • 15. adipogenic differentiation  maintained for 3 days in a-MEM (Gibco) supplemented with 10% FBS and antibiotics.  incubated in a-MEM supplemented with  10 mg/mLinsulin.  200 mM indomethacin.  0.1 mM dexamethasone.  0.5mM 3-isobutyl-1-methylxanthine .  10%FBS.
  • 16. neurogenic differentiation  neurobasal medium with neurogenic supplement.  10 ng/mL glial-derived neurotrophin factor.  10 ng/mL brain-derived neurotrophin factor.  10 ng/mL neurotrophin.  1 mL B-27 supplement.  2 mML-glutamine.  1% penicillin-streptomycin.
  • 17. RNA Extraction and RT-PCR of hTMSCs  Total RNA was extracted from the cells cultured differentiation usingTRIzol reagent .  Total RNA from each sponge- diluted to a volume of 10 mL in DEPC/DDW and used in a reaction (10 mL) containing  first-strand buffer ,  DTT  dNTP  oligo (dT) 15 primer  Rnase inhibitor  SuperScript II  Incubated at 45C for 60minutes.  The cDNA was stored at 220C until neede
  • 18. Amplification of dna  PCR reactions (50 mL) contained  Flexi DNApolymerase  MgCl2  dNTPs  cDNA template,  and primers specific : b-actin  aggrecan  type I collagen  type II collagen  osteocalcin  bone sialoprotein (BSP)  osterix  bone morphogenetic protein-2 (BMP-2)  The thermal conditions for PCR amplification were initially denaturation at 94C for 3 minutes, followed by 35 cycles of 94C for 30 seconds, 53C for 30 seconds, and 72C for 30 seconds,with a final extension at 72C for 5 minutes. PCR products were separated in 2% agarose (Sigma) gels in 13 Trisacetate- EDTA buffer containing ethidium bromide.
  • 19. Results
  • 20. Multilineage Differentiation Potential
  • 21. Expression of mRNA
  • 22.  Isolation of Novel Multipotent Neural Crest- Derived Stem Cells from Adult Human Inferior Turbinate  Stefan Hauser et al  STEM CELLS AND DEVELOPMENT Volume 21, Number 5, 2012
  • 23. HYPOTHESIS  The respiratory mucosa of adult human inferior turbinate may contain -NCSC population.
  • 24. Material and Methods  Cell isolation and culture of ITSCs  Biopsy tissues - immediately placed in PBS on ice- mechanical mincing using the Mcllwain TissueChopper.  Afterward, the tissue was dissociated using CollagenaseI.  The primary cultures were grown for at least 72h in serum-free medium consisting of Dulbecco’s modified Eagle’s medium/Ham F-12 (DMEM/F-12; Invitrogen) supplemented with  basic fibroblast growth factor-2  epidermal growth factor  Heparin For rapid expansion cultivated using DMEM/F-12 supplemented with: 10%human blood plasma 40 ng/mL FGF-2 20 ng/mL EGF.
  • 25. neuronal differentiation  For induced neuronal differentiation- re-suspended in DMEM containing  10% FBS  1 mM dexamethasone  2mM insulin  500mM 3-isobutyl-1-methylxanthine  200mMindomethacin  200mM ethanol  After 28 days, RNA isolation, immunocytochemical stainings and detection of synaptic vesicle .
  • 26. Adipogenic differentiation  For adipogenic differentiation, ITSCs - cultivated in  DMEM  10% FBS  1 mM dexamethasone  2mM insulin  500mM 3-isobutyl-1-methylxanthine  200mM indomethacin
  • 27. Chondrogenic differentiation  cultivated DMEM  10% FBS  10 ng/ml TGF-b1  0.1mM dexamethasone  Alcian blue - to detect the presence of glycosamino glycans
  • 28. Osteogenic differentiation  cultivated in DMEM  10% FBS  100 nM dexamethasone  10mM b-glycerophosphate  0.05mM L-ascorbic acid-2-phosphate  ALP activity was measured in differentiated ITSCs using the Alkaline Phosphatase Detection Kit  To detect biological mineralization cells were stained with Von Kossa and Alizarin Red S .
  • 29. Results  ITSCs - glial origin  Aarrowheads-p75 positive Schwann cell-like ITSCs  Arrows - axons  asterisk- Schwann cells
  • 30. ITSCs perform differentiation into ectodermal lineage  Cultivated in neuronal differentiation medium for 4 weeks-
  • 31. ITSCs are able to differentiate into representative mesodermal cell types
  • 32. Human Respiratory Epithelial Cells from Nasal Turbinate Expressed Stem Cell Genes Even after Serial Passaging  B H I Ruszymah et al  Med J Malaysia December 2011
  • 33. HYPOTHESIS  To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate.  FZD-9 and BST-1 were chosen as the stem cell marker
  • 34. MATERIALS AND METHODS  Six human nasal turbinate specimens under aseptic conditions, the specimens were digested in 0.3% Collagenase type I solution - 4-12 hours.  The cell suspension containing fibroblasts and respiratory epithelial cells - centrifuged at 6,500 rpm - 5 minutes.  resuspended in 5-10ml trypsin EDTA and kept in shaker incubator - 5 minutes at 370C.  same volume of Trypsin Inhibitor (Gibco/BRL, USA)  cultured - Defined Keratinocytes Serum Free Medium (DKSFM) +Modified Eagle’s Medium (DMEM) supplemented with 5% Fetal Bovine Serum (FBS)
  • 35. RESULTS
  • 36. Discussion  The lamina propria—main constituent loose connective tissue rich network of thin-walled venous sinusoids fibroblasts, macrophages, T and B lymphocytes, plasma cell
  • 37.  All 3 study strongly suggest that these cells are MSCs and demonstrate the possibility of using human inferior turbinate tissues removed during turbinate surgery as a source of stem cells  In culture, even after multiple passage no change in characteristic of these cells.
  • 38.  The ability to easily isolate MSCs - tissues discarded during surgical procedures  reduce pain and morbidity in MSC donors increase donor participation  In all avobe 3 study dicarded tissue used for cultivation of MSCs.
  • 39.  Corporation of Korea--- 134,619 turbinate surgeries performed in 2009 to treat enlarged inferior turbinates.  Sufficient turbinate tissue should be available to support seed cell or individual cell banks.  Future use of tissue that is normally discarded during surgery may enable the establishment of custom-made tissue and cell banks for patients.
  • 40. Limitation To compare proliferation and differentiation between hTMSCs and MSCs from bone marrow and adipose tissue.  bone marrow–derived MSCs exhibit an age-related decline in life span, proliferation, and osteogenic potential-our study did not explianed about this.  No compare of hypertrophic turbinate - with normal turbinate tissue.
  • 41. The Human Nose Harbors a Niche of Olfactory Ectomesenchymal Stem Cells Displaying Neurogenic and Osteogenic Properties STEM CELLS AND DEVELOPMENT
  • 42. Conclusion  Above studies proved that hTMSCs can differentiate into other types of adult cells.  This will allow us to develop an effective tissue-regenerating method with the use of adult cells.  Cell banks can be established from stromal-cell-rich turbinate tissues that would otherwise be discarded after nasal surgery.

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