Name: isolation and analysis of hematopoietic stem cells from the placenta
Uploaded: 2008-06-24T16:25:00
Description: Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

1. Embryo removal and placental dissection
<ol>
<li>To begin, embryos must first be isolated from pregnant dams. </li>
<li>Animals are euthanized according to approved procedures &ndash; in our case we are going to use a lethal dose of the anaesthetic isoflurane</li>
<li>First, spray the belly with ethanol and make a small incision with a pair of scissors.</li>
<li>Tear away the skin with both hands to uncover the abdomen and cut open the peritoneum. </li>
<li>With forceps, pick up the uterine horns and collect them usi...

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

The experimental procedures described in this protocol will allow for isolation and visualization of placental tissue and hematopoietic stem and progenitor cells. For a comprehensive summary on expected cellular yield and number of HSCs in placenta and other fetal hematopoietic organs throughout fetal development we refer to Gekas, et al. 2005. For localization of developing HSCs and other hematopoietic cells in the placenta we refer to Rhodes, et al. 2008.

Materials List: 
100mM Glucose 
10mM NaN3 
15-ml conical tubes (BD) 
16-G, 18-G, 20-G, 22-G and 25-G needles (BD) 
30% Sucrose Solution 
35mm Petri Dishes (BD) 
4% Paraformaldehyde 
50- m cell filters (Celltrics) 
5-ml syringes (BD) 
Alkaline Phosphatase Substrate Kit I (Vector Red, Vector) 
Alkaline Phosphatase Substrate Kit III (Vector Blue, Vector) 
Antibiotic (Penicillin/Streptomycin, P/S) solution 
Collagenase type I (Sigma) 
Cryomold Disposable Vinyl Specimen Molds (10mm x 10mm x 5 mm, Tissue-Tek) 
Cytokeratin Primary Antibody (DakoCytomation) 
Dissection forceps, stainless steel or titanium, number 5 or 55 
Drierite 
Fetal Bovine Serum (FBS) 
Fisherbrand Plastic Microscope Slide Mailer (Fisher) 
Glucose Oxidase 1000u/ml 
Levamisole (Vector) 
Mounting media, Aqueous-based Mounting Medium (VectamountTM AQ) 
Normal Horse Serum (Vector) 
Optimal Cutting Temperature (O.C.T., Tissue-Tek) 
Peroxidase Substrate Kit DAB (Vector) 
Phosphate Buffered Saline (PBS) with Calcium/Magnesium (w Ca2+/Mg2+) 
Phosphate Buffered Saline (PBS) without Calcium/Magnesium (w/o Ca2+/Mg2+) 
Plastic Slide Box (holds 100 slides) 
Plastic Tubing (Nalgene 180 PVC Non-toxic Autoclavable) 
Polypropylene Round-Bottom Test Tube, 5 ml (12x75 mm, BD Falcon) 
Proteinase K 20mg/ml Solution (Amresco) 
Research Products International Corp Super Pap Pen HT* Slide Markers (2,5 mm, Fisher) 
Tissue-culture treated U-shaped (round-bottom) 96-well or 24-well plates (BD Falcon) 
Tween20, SigmaUltra (Sigma) 
Tyramide Amplification Kit (Invitrogen) 
Vectastain ABC Alkaline Phosphatase Standard Kit (Vector) 
Vectastain ABC Standard Elite Kit (Vector)

isolation and analysis of hematopoietic stem cells from the placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...