CMRC iPS and Human Stem Cell Core Facility

Vasil Galat, Ph.D. , Director 
Philip Iannaccone, M.D., Ph.D., co-Director
 

Cover stories: 

 JCP cover Cover shows the differential expression of microRNAs (miRs) in human embryonic stem cells (hESCs) vs. human-induced pluripotent stem cells (hiPSCs), revealing 10 highly expressed miRs in hiPSCs with greater than ten-fold difference, which have been shown to be cancer related.
(From: Malchenko S, V. Galat, E. A. Seftor, E. F. Vanin, R. E.B. Seftor, M. B. Soares and M. J.C. Hendrix. Cancer hallmarks in induced pluripotent cells: new insights. Journal of Cellular Physiology. 2010. 225; 2; 390-393. PMID: 20568225) 
    SCD cover The panel illustrates the developmental potential of novel rat embryonic stem cells with extraembryonic commitment. Shown: the green fluorescent protein expressing stem cells integrated into yolk sac, trophectoderm or induced a blastocoel formation upon injection into morula stage rat and mouse embryos.
(From: Galat V, Bert Binas, Stephen Iannaccone, Lynne-Marie Postovit, Bisrat Debeb and Philip Iannaccone. Developmental potential of rat extraembryonic stem cells. Stem Cells and Development. 2009. 9. 1309-18. PMID: 19480599)

 

Current repository of hESC lines

1) Established in Children's Memorial Research Center: CM 1; CM 2; CM 3; CM 4; CM 5; CM 6; CM 7; CM 8; CM 9; CM 10; CM 11; CM 12; CM 13; CM 14; and CM 16.

2) Acquired from a National Stem Cell Bank (NIH approved): H1; H7; H9; H13.B; and H14.
 

Current repository of induced pluripotent stem cells (iPSC) established in Children's Memorial Research Center 

1) iPSC line reprogrammed from fibroblasts (ATCC, MRC-5)

2) iPSC line reprogrammed from fibroblasts (Coriell, AG08642, trisomy 21)
 

hESC on MEF  hESC on matrigel 
 Morphology of hESC (H9) growing on MEF (left) and matrigel (right)

 What does the Stem Cell Core Facility do? 

 
  • Promotion of collaborative studies in stem cell biology 
  • Focus on genetic regulations of stem cell lineages, directed differentiation and utilization for therapy 
  • Study of human diseases using hESCs and iPSCs with genetic genetic mutations 
  • Development of repository of hESCs and iPSCs 

What is a stem cell?

A stem cell is an undifferentiated cell that can both renew itself (divide for indefinite periods in culture) and give rise to specialized cells (differentiate). 
Embryonic Stem Cells are isolated from preimplantation stages, that is, before the embryo attaches to the uterine wall and before fetal development begins. Typically this is done at the blastocyst stage of development when the embryo is a hollow ball of cells. These blastocysts are placed in a dish and the stem cells grow out of them, creating a dish filled with individual cells that have the capacity to differentiate into all cell types.  

Advantages:

  • Make exact copies of themselves  
  • Multiply rapidly  
  • Totipotent/Pluripotent  

Disadvantages:

  • Ethical considerations  

Embryo donation is the best understood method of obtaining new human embryonic stem cells. This is done with discarded in vitro fertilization (IVF) embryos. All IVF clinics produce more embryos than can be used and for most couples these extra embryos are frozen in case they are needed. At some point the infertility treatment succeeds and the parents are finished with the embryos. They can be maintained in the frozen state but over time the ability of these embryos to develop normally is lost and eventually they die. Parents are often anxious to have these embryos serve an important altruistic purpose and chief among these is stem cell research.

Adult stem cells are typically isolated from bone marrow or other tissues. They have the advantages of being from the patient potentially (and so can be transplanted easily) and eliminate the ethical considerations of isolating cells from embryos. However, they have limited developmental potential and limited life spans. If the patient has a genetic disease then the patient’s own stem cells will not be helpful.

Stem cells can also be derived from embryos that have undergone preimplantation genetic diagnosis. These embryos have undergone biopsy to determine if a genetic disorder exists and they can be used to derive embryonic stem cells. This approach can yield stem cells that have the genetic disorder, not to be use for therapy but to study the disease.

Another technique for generating sources of stem cells is somatic cell nuclear transfer (SCNT), or “therapeutic cloning.” This technique is considered by some to be most acceptable as no fertilized embryo is used. Also the stem cells derived are the same genetically as the donor, so where this is appropriate transplant of the cells is easier. This technique involves transferring the nucleus, and therefore the DNA, of a somatic cell (a body cell, typically skin or muscle) of a donor to an egg that has had its own nucleus removed. An alternative method involves a process known as parthenogenesis where the egg divides on its own although it never got fertilized, which is observed naturally in many different species including plants, insects, as well as in lizards and other vertebrates. These eggs are then used as the source for stem cells.

In SCNT, after the somatic cell nucleus has been inserted into the enucleated egg, it is stimulated to encourage it to begin dividing on its own either chemically or by electrical impulses. After many divisions have occurred in culture, the egg forms a blastocyst, which is a ready supply of competent stem cells. Because the nuclei of the dividing cell was taken from the somatic cells of a donor, the resulting stem cells are genetically matched to that donor, which makes SCNT an exciting area of research for therapeutic discoveries. If these stem cells are used to develop tissues to help or replace those damaged in the donor, brain cells in patients of Parkinson’s disease, islet cells in diabetes, retinal pigment epithelial (RPE) cells for retinal degeneration (Lund et al., 2006), or in cases of spinal cord injury for example, there is no more need to suppress the immune system of the recipient for fear of transplant rejection. SCNT offers possibilities of genetically tailoring therapies to an individual’s disease. For this research to continue moving forward, sources of stem cells are needed. We would like to use well established techniques for nuclear transfer and parthenogenesis that we and others have used in animal models with human eggs to develop this important technique for stem cell therapy.

Adult cells can be induced to behave like embryo derived stem cells by transfecting or transducing the expression of several critical genes producing so-called iPS cells. This approach offers the opportunity to derive histocompatible stem cells without the disruption of embryos. The disadvantage is the unknown effects of the genetic manipulation and the expression of potential oncogenes in cells intended for therapeutic uses.

What is the promise of stem cells?

There is mounting evidence that stem cells isolated from adult tissues, human embryos at blastocysts stage, cord blood and placenta have potential to correct experimental disease models such as Neurological Diseases, Juvenile Diabetes, Spinal Cord Injuries, Heart Defects, Muscle Defects, and rescue of pigmented retinal epithelium.