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 of Biotechnology, JMIT, Radaur, Haryana, India 2 NIDDK, National Institutes of Health, Bethesda, MD 20892, USA Corresponding:

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”.