Journal Sanjaya

  • View
    226

  • Download
    0

Embed Size (px)

Text of Journal Sanjaya

  • 7/31/2019 Journal Sanjaya

    1/15

    HUMAN BREAST MILK IS A RICH

    SOURCE OF MULTIPOTENTMESENCHYMAL STEM CELLS

    1

    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

  • 7/31/2019 Journal Sanjaya

    2/15

    INTRODUCTION

    Saturday , January 08, 20112

    A stem cell is a cell that has the ability to divide for indefiniteperiods in culture.

    On the basis of developmental potential, four levels of stemcells can be recognized: totipotent,pluripotent,multipotent, andunipotent.

    Mesenchymal stem cells(MSCs) are the cells from immatureembryonic connective tissue.

    MSCs have been isolated from bone marrow, adipose tissue,tendon, periodontal ligament, synovial membrane and lungs,including tissue fluids such as synovial fluid, amniotic fluid andmenstrual blood etc.

  • 7/31/2019 Journal Sanjaya

    3/15

    Saturday , January 08, 20113

  • 7/31/2019 Journal Sanjaya

    4/15

    OBJECTIVES

    Saturday , January 08,20114

    To isolate and expand a mesenchymal stem cell-likepopulation from human breast milk.

    To examine cultured cells by immunofluorescent labeling andcytoskeleton protein marker.

    To test the multipotent differentiation potential of the stem cell.

  • 7/31/2019 Journal Sanjaya

    5/15

    MATERIALS AND METHODS

    Saturday , January 08, 20115

    COLLECTIONOF BREASTMILK SAMPLE

    With their written consent, breast milk samples were

    collected from 35 healthy volunteers.

    The samples were collected in sterile 15ml Falcon tubes

    and transported to the laboratory for isolation.

  • 7/31/2019 Journal Sanjaya

    6/15

    ISOLATION & CULTURE OF CELLS

    Saturday , January 08, 20116

    Every 48 hrs medium was changed and replenished(passage).

    At 80% confluency,cells were passage using Trypsin EDTA

    Cell pellet was washed twice with sterile phosphate buffered saline(PBS)

    Cell pellet was seeded in a tissue culture dish containing 10% hUCBS.

    Breast milk was diluted 1:2 with Dulbecco's modified eagle medium(DMEM) containing antibiotics.

    Centrifuged at 285g for 10 m

  • 7/31/2019 Journal Sanjaya

    7/15

    CHARACTERIZATION OF ISOLATED CELLS

    Cover slips were mounted on medium containing antifade &4,6-diamino-2-phenylindole(DAPI) for nuclear visualization.

    Then exposed to primary antibodies namely nestin,vimentin,smoothmuscle actin(SMA) and E-Cadherin for 12 hrs at 4c

    Exposed with secondary antibodies for 1 hr at 37c.

    Cells were fixed with 4% paraformaldehyde, then permiabilized using50% methanol for 5 min.

    Mixed with 5% bovine serum albumin(BSA) in PBS for 1 hr.

    Saturday , January 08, 20117

  • 7/31/2019 Journal Sanjaya

    8/15

    Saturday , January 08, 20118

    The cells were fixed in 70% ethanol then incubated in fluoresceinisothiocyanate cojugated antibodies against

    CD33,CD34,CD44,CD45,CD73,CD90 and CD117 for 1 hr on ice.

    The cells were acquired using a flow cytometer laser 488nm and datawere analysed using BD cellquest pro software.

  • 7/31/2019 Journal Sanjaya

    9/15

    Saturday , January 08, 20119

    MSCs were introduced in the alternatecycle of adipogenic induction andadipogenic maintenance medium.

    Adipogenesis was confirmed using oil Red Ostaining

    Similarly with differentiation bulletin kit Chondrogenesis &Osteogenesis were confirmed using Safranin-O & AlizarinRed S staining respectively.

    DIFFERENTIAL STUDIES

  • 7/31/2019 Journal Sanjaya

    10/15

    RESULT

    Saturday , January 08, 201110

    Initially, the isolatedcell showed an

    epithelial-like cell

    population.

    During second week,cell resembled atypical slender

    fibroblast-like cell

    phenotype.

  • 7/31/2019 Journal Sanjaya

    11/15

    RESULT..

    The isolated human breast milk cells expressed mesenchymal markers

    namely SMA, Vimentin,Nestin and surface markers CD44,CD29,SCA1.

    The Confocal microscopic study showed high level of expression of

    epithelial marker E-Cadherin along with nestin & vimentin.

    The cells were found to be negative for CD33,CD34,CD45,CD73 by

    fluorescence activated cell sorting(FACS),confirming their identity as

    MSCs.

    Saturday , January 08,201111

  • 7/31/2019 Journal Sanjaya

    12/15

    Due to lack of specific known markers, differentiation potential

    was studied with further incubation of 21 days.

    Saturday , January 08, 201112

    Spindle-shaped cells round oval cells

    Intra-cytoplasmic liquid droplets stained

    positive with Oil Red O.

    Adipogenic

    differentiation

    Spindle-shaped cells large round cells Sulphated proteoglycans stained

    positive with safranin O.

    Chondrogenicdifferentiation

    Spindle-shaped cells cuboidal cells

    Calcium phosphate stained positive withAlzarin red S stain.

    Osteogenicdifferentiation

  • 7/31/2019 Journal Sanjaya

    13/15

    CONCLUSION

    Human breast milk cells display stem cell properties and are capable of

    differentiating into various lineages.

    MSCs isolated from human breast milk could potentially be reprogrammed

    to form many types of human tissues.

    Lastly, MSCs could be useful in treatment of spinal injuries, diabetes and

    Parkinson's diseases.

    Saturday , January 08, 201113

  • 7/31/2019 Journal Sanjaya

    14/15

    CRITICS

    Further determination of the significance of these cells is required.

    Saturday , January 08, 201114

  • 7/31/2019 Journal Sanjaya

    15/15

    Saturday , January 08, 201115