Journal Sanjaya

7/31/2019 Journal Sanjaya

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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


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INTRODUCTION


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.


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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.


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MATERIALS AND METHODS


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.


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ISOLATION & CULTURE OF CELLS


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


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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.



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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.


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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


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RESULT


Initially, the isolatedcell showed an

epithelial-like cell

population.

During second week,cell resembled atypical 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.

Saturday , January 08,201111


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Due to lack of specific known markers, differentiation potential

was studied with further incubation of 21 days.


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


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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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