Your stem cell company had been working with two patients with heart disease to
ID: 64813 • Letter: Y
Question
Your stem cell company had been working with two patients with heart disease to develop iPS cells from each patient to be used for treatment. Skin cells were taken from each patient, transformed using the standard factors, and iPS cells were selected and grown in culture in preparation for treating the patients. The hope was that infusing iPS cells derived from the patient’s own skin cells would repair the heart damage without triggering an immune response. However, in the natural disaster that damaged the hospital (mentioned in problem 4, shown below) the labels of the two cell cultures that identified which culture belonged to which patient were erased, although the cells are still viable.
Problem 4 ----A major earthquake has damaged a hospital and all records were lost. One infant was rescued and two different sets of relatives (aunts, uncles, and grandparents) are both claiming this child is their relative. Both families are certain their family’s child was conceived by artificial insemination, but in both cases the parents were lost. One family knows that their child had three biological parents (and they know the mitochondrial donor)
a. What test or tests would you do to determine which culture of cells belongs to which patient?
b. What results would you expect from your tests that would confirm the cells identity
Explanation / Answer
Identifying the iPSCs accurately is somewhat difficult at the time of somatic cell reprogramming. As the cell reprogramming is a complex multistep process that takes a few weeks to get completed, identifying the exact pluripotent colonies in the culture of transformed fibroblasts and other cell debris might be an easy task for an experienced person. Identification can be a challenging one for those who do not have desired experience in this field.
a. There is a test called Alkaline phosphatase live stain to identify the specific iPSCs. The candidate iPSC colonies are examined for the expression of pluripotent markers making use of immuno-cytochemical analyses. A common method of identifying the specific iPSC colonies is by using stem cell specific antibodies that can distinguish proteins like SSEA4, TRA-1-80 and TRA-1-60. These proteins are expressed highly on the surface of human stem cells.
However, this method can be employed after the colonies emerge and cannot be utilized in the early reprogramming process. So, colonies cannot be distinguished on the master plate. Alkaline phosphatase activity is usually used to monitor and identify stem cells from the feeder cells and parental cells in the reprogramming experiments.
Apart from the antibody based analyses alkaline phosphatase substrates are used for early screening of the stem cells. This substance is mostly toxic to the cells and therefore needs endpoint monitoring.
Other than traditional alkaline phosphatase stains, the molecular probes of the live stain can be used to label the pluripotent stem cells when it is enzymatically induced. Alkaline phosphatase stain can produce a dark green fluorescent product that helps to identify the emerging iPSCs.
The pluripotency of the stem cells can be confirmed by using a test that includes stem cell specific antibodies for the membrane surface markers TRA-1-81 and SSEA4 proteins.
b. The alkaline phosphatase live staining of the mitochondrial donor stem cells and that of the two stem cell cultures of the patients can be done to compare the results. Antibody specificity of the semen donor can be compared with that of the two child cultures to determine the parental origin. Therefore, the family that knows about the artificial insemination for the birth of their child by a mitochondrial donor can claim that the child belongs to that family. The other family which simply knows about artificial insemination and do not know about the relation between the mitochondrial donor and the child may not own that child.
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