HUMAN BREAST MILK IS A RICH SOURCE OF MULTIPOTENT MESENCHYMAL STEM CELLS

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

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INTRODUCTION

A stem cell is a cell that has the ability to divide for indefinite periods in culture. On the basis of developmental potential, four levels of stem cells can be recognized: totipotent,pluripotent,multipotent, and unipotent. Mesenchymal stem cells(MSCs) are the cells from immature embryonic 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 and menstrual blood etc.
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OBJECTIVES

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

To examine cultured cells by immunofluorescent labeling and cytoskeleton protein marker.

To test the multipotent differentiation potential of the stem cell.

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MATERIALS AND METHODS

COLLECTION OF BREAST MILK 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.

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ISOLATION & CULTURE OF CELLS

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

Every 48 hrs medium was changed and replenished(passage). At 80% confluency,cells were passage using Trypsin EDTA

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CHARACTERIZATION OF ISOLATED CELLS

Cells were fixed with 4% paraformaldehyde, then permiabilized using 50% methanol for 5 min. Mixed with 5% bovine serum albumin(BSA) in PBS for 1 hr. Then exposed to primary antibodies namely nestin,vimentin,smooth muscle actin(SMA) and E-Cadherin for 12 hrs at 4c Exposed with secondary antibodies for 1 hr at 37c.

Cover slips were mounted on medium containing antifade &4,6-diamino2-phenylindole(DAPI) for nuclear visualization.

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The cells were fixed in 70% ethanol then incubated in fluorescein isothiocyanate 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 data were analysed using BD cellquest pro software.

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DIFFERENTIAL STUDIES

MSCs were introduced in the alternate cycle of adipogenic induction and adipogenic maintenance medium.

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.

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RESULT

Initially, the isolated cell showed an epithelial-like cell population.

During second week, cell resembled a typical slender fibroblast-like cell phenotype.

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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.
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Due to lack of specific known markers, differentiation potential was studied with further incubation of 21 days.

Adipogenic differentiation Chondrogenic differentiation Osteogenic differentiation

Spindle-shaped cells round oval cells Intra-cytoplasmic liquid droplets stained positive with Oil Red O. Spindle-shaped cells large round cells

Sulphated proteoglycans stained positive with safranin O. Spindle-shaped cells cuboidal cells Calcium phosphate stained positive with Alzarin red S stain.

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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.

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CRITICS

Further determination of the significance of these cells is required.

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