A novel source for Mesenchymal stem cells Jayanti Tokas 1, Deepika Gupta 1, Divya Pasrija 1, Rubina Begum 1, Shalini Jain 2 and Hariom Yadav 2 1 Department.

A novel source for Mesenchymal stem cells

Jayanti Tokas1, Deepika Gupta1, Divya Pasrija1, Rubina Begum1, Shalini Jain2 and Hariom Yadav2

1Department of Biotechnology, JMIT, Radaur, Haryana, India

2NIDDK, National Institutes of Health, Bethesda, MD 20892, USA

Corresponding: [email protected]

FETAL STEM CELLS

Sources Specific fetal tissues Fetal circulation, placenta, Amniotic fluid Umbilical cord

Potentiality varies from pluripotent to multipotent.

Fetal stem cells Hemopoietic stem cells (HSC) Mesenchymal stem cells (MSC)

First-trimester blood richer source of HSC and MSC Greater proliferative capacity wider differentiation ability

Can be clonally derived.

FETAL STEM CELLS

AMNIOTIC FLUID

Clear, yellowish liquid, surrounds the fetus during pregnancy

Continuously inhaled, exhaled and urinated by the baby.

It is 98% water and electrolytes, proteins, peptides, carbohydrates, lipids and hormones.

Protects the developing baby against blows to the mother's abdomen

Allows easier fetal movement

Promotes muscular/skeletal development

Protect the fetus from heat loss.

Plays a significant defensive role

AMNIOTIC FLUID

Amniotic fluid

ROLE IN FETAL DEVELOPMENT

Contains nutritional components such as proteins, glucose, triglyceride and cholesterol

proteins and peptides possess potent bioactivity

Growth factors in AF are transported throughout fetal body

Helps cellular movement, organs development, cellular growth and proliferation.

AMNIOTIC FLUID STEM CELLS

Potentiality varies from pluripotent to multipotent

Second and third trimester amniotic fluid is major source

Allow scientists to sidestep the controversy over destroying embryos for research

Can be used for tissue repair and engineering organs

Mesenchymal stem cells quantity is high in amniotic fluid

HETEROGENEITY OF AMNIOTIC FLUID

Variation in Shape Size Nuclear/ cytoplasmic ratio Cytoplasmic characteristics Cell surface properties Biochemical properties

Some cells express markers of glial and neuronal stem cells.

AMNIOTIC FLUID :NEW SOURCE FOR NEURAL STEM CELLS

Amniotic fluid cells when incubated with neurogenic medium express

CD133 Nestin Neurofilament CNPase p75 Neurotrophin receptor (the brain-derived neurotrophic factor and

neurotrophin-3)

Human amniotic fluid contains cells that express markers for neuronal stem and progenitor cells, which harbour the potential to differentiate

into neurogenic cells

MESENCHYMAL STEM CELLS

Morphology

Small cell body with a few cell processes Large and round nucleus with a prominent

nucleolus A small amount of

Golgi apparatus Rough endoplasmic reticulum Mitochondria Polyribosomes .

Detection Surface antigens

Differentiation capacity Multipotent- Can differentiate into

Osteoblasts Adipocytes chondrocytes myocytes neuron-like cells.

Differentiation capacity decreases with the age of the donor and the time in culture

MESENCHYMAL STEM CELLS

MESENCHYMAL STEM CELLS AND ITS DIFFERENTIATION CAPACITY

ISOLATION AND CULTURE

Sterile needle is inserted through the abdominal wall into the amniotic sac

Small amount of amniotic fluid is withdrawn through needle

Amniotic fluid contains fetal cells which are separated from amniotic fluid

Cultured

Tests are performed on cultured cells Chemical analysis

DNA analysis Chromosomal Analysis

MESENCHYMAL STEM CELLS : FIRST TRIMESTER

CD291

CD441

SH21

SH31

SH41

prolyl-4-hydroxylase

Actin

CD452

CD342

CD142

CD682

Vwf2

HLA-DR2

Fibronectin

Laminin

Vimentin

In undifferentiated state, fetal blood, liver and bone marrow MSCs express

MSCs differentiate into Adipocytes Osteocytes Chondrocytes.

Provide novel targets for in utero cellular and gene therapy.

MESENCHYMAL STEM CELLS : FIRST TRIMESTER

MSCs exhibit a phenotype and multilineage differentiation potential similar to postnatal bone marrow (BM) – derived MSCs .

Multipotent (CD29)

Pluripotent (Oct-4)

MESENCHYMAL STEM CELLS : SECOND TRIMESTER

Express Oct-4.

Samples can be collected in larger amount than the second trimester, with a much lower risk of uterine contamination.

High renewal capacity and therapeutic applications.

Share same properties and differentiation capabilities as human embryonic stem cells .

Differ from embryonic stem cells in promising ways :

1. Do not produce teratomas when transplanted into animals.

2. Low antigenicity is an advantage for cell transplantation or cell replacement therapy.

MESENCHYMAL STEM CELLS : THIRD TRIMESTER

PRESERVATION

Vitrification

Amniotic stem cell banks

VITRIFICATION

AMNIOTIC STEM CELL BANKS

Biocell center, European biotechnology company is the first firm to harvest and preserve amniotic stem cells

Biocell Center captures the stem cells from amniotic fluid if the family requests and pays for the preservation of the stem cells

Amniotic fluid withdrawn during amniocentesis is sent to the Biocell Center laboratory. The stem cells are frozen in liquid nitrogen and preserved in the company's state-of-the-art cryo-bank.

“H.R. 1892—National Amniotic and Placental Stem Cell Bank of 2007

The bill shows the importance of amniotic stem cells bank

which would

Establish a National Amniotic and Placental Stem Cell Bank for the purpose of obtaining, storing, and making available for research and treatment human stem cells derived from amniotic fluid or placenta .

Will maintain a collection of at least 100,000 samples of stem cells in order to ensure genetic diversity

Will obtain stem cells only if informed consent is provided

REGENERATIVE MEDICINE

As regenerative medicine amniotic fluid stem cells has many applications .

Cultured MSCs have been transplanted in children with osteogenesis imperfecta (OI), a disease causing bone fractures and fragility.

Reduced bone fractures and increased bone density were reported to be found when MSCs were engrafted into the defective bone

MYOCARDIAL INFARCTION

MSCs can be engrafted at the damaged

site(s), attenuate pathologic remodeling of

the heart tissue and reduce scar size,

leading to improved post-MI cardiac

function

ETHICAL ASPECTS

Amniotic fluid solve a lot of problems

It's possible to catch amniotic stem cells without destroying embryos

They are not necessary to choose an alternative between donor or autologuous use

NEXT STEPS TO DO

For the future it is of great importance to obtain more insights into the spectrum of cells contained in human amniotic fluid.

Using RT-PCR the expression of a wide variety of genes, known as markers for stem cells, progenitor cells and differentiated cell types, should be analysed.

Amniotic fluid contains Oct-4 positive cells, a major issue for the future is to investigate the differentiation potential of the Oct-4 positive amniotic fluid cells

It is essential to think about strategies to isolate/enrich Oct-4 positive cells out of amniotic fluid.

“It is an ocean full of pearls

and we need to search

these pearls only for our

benefit”.