Satish PATKI, Sachin KADAM, Vikash CHANDRA and Ramesh BHONDE Patki research foundation and hospital, shahupuri, kolhapur, National center for cell science,Ganeshkhind, pune, india.

Sanjaya Kumar Pant Roll No : 34 MSc Microbiology National College, Kathmandu


Saturday , January 08, 2011

Saturday . Mesenchymal stem cells(MSCs) are the cells from immature embryonic connective tissue. January 08. and unipotent. adipose tissue.pluripotent. On the basis of developmental potential. four levels of stem cells can be recognized: totipotent. 2011 2 .INTRODUCTION     A stem cell is a cell that has the ability to divide for indefinite periods in culture. including tissue fluids such as synovial fluid.multipotent. tendon. periodontal ligament. amniotic fluid and menstrual blood etc. synovial membrane and lungs. MSCs have been isolated from bone marrow.

January 08.3 Saturday . 2011 .

OBJECTIVES  To isolate and expand a mesenchymal stem cell-like population from human breast milk. 2011 .  4 Saturday . January 08.  To examine cultured cells by immunofluorescent labeling and cytoskeleton protein marker. To test the multipotent differentiation potential of the stem cell.

5 Saturday . • The samples were collected in sterile 15ml Falcon tubes and transported to the laboratory for isolation.MATERIALS AND METHODS COLLECTION OF BREAST MILK SAMPLE • With their written consent. breast milk samples were collected from 35 healthy volunteers. 2011 . January 08.

2011 . January 08. Every 48 hrs medium was changed and replenished(passage).ISOLATION & CULTURE OF CELLS Breast milk was diluted 1:2 with Dulbecco's modified eagle medium (DMEM) containing antibiotics. At 80% confluency. Centrifuged at 285g for 10 m Cell pellet was washed twice with sterile phosphate buffered saline(PBS) Cell pellet was seeded in a tissue culture dish containing 10% hUCBS.cells were passage using Trypsin EDTA 6 Saturday .

6-diamino2-phenylindole(DAPI) for nuclear visualization. then permiabilized using 50% methanol for 5 min.CHARACTERIZATION OF ISOLATED CELLS Cells were fixed with 4% paraformaldehyde.smooth muscle actin(SMA) and E-Cadherin for 12 hrs at 4c Exposed with secondary antibodies for 1 hr at 37c. 7 Saturday . January 08. Cover slips were mounted on medium containing antifade &4.vimentin. 2011 . Then exposed to primary antibodies namely nestin. Mixed with 5% bovine serum albumin(BSA) in PBS for 1 hr.

January 08.CD90 and CD117 for 1 hr on ice.The cells were fixed in 70% ethanol then incubated in fluorescein isothiocyanate cojugated antibodies against CD33. 2011 .CD45. The cells were acquired using a flow cytometer laser 488nm and data were analysed using BD cellquest pro software.CD44. 8 Saturday .CD34.CD73.

Adipogenesis was confirmed using oil Red O staining Similarly with differentiation bulletin kit Chondrogenesis & Osteogenesis were confirmed using Safranin-O & Alizarin Red S staining respectively.DIFFERENTIAL STUDIES MSCs were introduced in the alternate cycle of adipogenic induction and adipogenic maintenance medium. 9 Saturday . January 08. 2011 .

cell resembled a typical slender fibroblast-like cell phenotype. During second week.RESULT Initially. the isolated cell showed an epithelial-like cell population. January 08. 2011 . 10 Saturday .

confirming their identity as MSCs.CD73 by fluorescence activated cell sorting(FACS). January 08. Saturday .CD45. • The Confocal microscopic study showed high level of expression of epithelial marker E-Cadherin along with nestin & vimentin.CD29. • The cells were found to be negative for CD33.RESULT….Nestin and surface markers CD44.CD34. 2011 11 . • The isolated human breast milk cells expressed mesenchymal markers namely SMA. Vimentin..SCA1.

January 08. 12 Saturday . Adipogenic differentiation Chondrogenic differentiation Osteogenic differentiation • Spindle-shaped cells round oval cells • Intra-cytoplasmic liquid droplets stained positive with Oil Red O.Due to lack of specific known markers. 2011 . differentiation potential was studied with further incubation of 21 days. • Spindle-shaped cells cuboidal cells • Calcium phosphate stained positive with Alzarin red S stain. • Spindle-shaped cells large round cells • Sulphated proteoglycans stained positive with safranin O.

CONCLUSION • Human breast milk cells display stem cell properties and are capable of differentiating into various lineages. diabetes and Parkinson's diseases. MSCs could be useful in treatment of spinal injuries. • • 13 Saturday . 2011 . MSCs isolated from human breast milk could potentially be reprogrammed to form many types of human tissues. January 08. Lastly.

2011 .CRITICS • Further determination of the significance of these cells is required. 14 Saturday . January 08.

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