Q and A follow-up: Panel discussion on stem cell characterization

Follow-up of the live panel discussion Q and A session.

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Jan 23, 2018
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Thank you to everyone who attended the live panel discussion on stem cell characterization. Below are the responses to the questions posed during the live event that we did not have time to answer. We hope this is a useful resource and thank our attendees and our speakers, James L. Sherley (Asymmetrex, LLC), Glynn Stacey (International Stem Cell Banking Initiative)and Rouzbeh R. Taghizadeh (AuxoCell), for their time.

If you missed this event, you can still watch the panel discussion on demand.

Does adherent versus suspension culture of tissue stem cells affect their health and quality?

James: The answer to this question depends on the specific requirements of stem cells from different tissues. Generally, cells that require adherence do not do well when cultured without attachment.  Some types of stem cells, like hematopoietic stem cells, do not appear to require attachment naturally.  Many other stem cells from epithelial tissues and neural tissues seem to have the ability to form and survive in cell microspheres in non-adherent culture. Attachment requirements seem to be provided by differentiating cells that also compose the microspheres. In the past, microsphere formation has been considered as a common property of adult tissue stem cells

Does the specific counting issue impact basic tissue stem cell research as well? 

James: Absolutely. Currently much of basic tissue stem cell research is conducted without knowing the number of adult tissue stem cells in experiments. This is certainly usually the case for stem cells from epithelial and mesenchymal tissues. Some possible exceptions are studies with muscle satellite stem cells, for which the combination of Pox 7 expression, asymmetric division, and migration properties may specifically identify the cells; and studies using detection of non-random chromosome co-segregation to detect and quantify asymmetrically self-renewing tissue stem cells. Similarly, time-lapse microscopy may be used to estimate the frequency of asymmetrically self-renewing tissue stem cells in culture. In the special case of hematopoietic stem cells (HSCs), limiting dilution reconstitutions in immune-deficient mice gives an estimate of HSC number. However, in reality, the number estimated can be confounded by differences in the engraftment efficiency of the cells.

Are they any types of tissue stem cells that are not diluted during expansion in culture?                            

James: Cancer stem cells that have lost the property of asymmetric self-renewal and have generation rates that approach or exceed the rates of other cell types in the culture will not undergo dilution. In fact, the loss of asymmetric self-renewal by these cells has been postulated as the basis for their immortalized properties. Any tissue stem cell type that divides asymmetrically sufficiently more frequently than symmetrically will eventually dilute to a level approaching 0%. If a stem cell type can be maintained instead with a sufficiently greater frequency of symmetric divisions, it will be possible to maintain and increase its fraction and total number in culture. For therapeutic applications of tissue stem cells maintained and expanded in this manner, the increase in symmetric division frequency must be reversible, so that normal tissue cell renewal can be regained and carcinogenesis avoided.

Why PBSC cannot be cultured for stem cell transplantation of lymphoma or myeloma patient? 

James: PBSCs (peripheral blood stem cells) are hematopoietic stem cells that also divide primarily asymmetrically and are still quite rare even in growth factor-mobilized populations. Their differentiation lineages are particularly prolific for cell proliferation. This combination of properties is predicted to result in the most rapid dilution of PBSCs when cultured. There may be other factors as well, like cell death and apoptosis that will serve to further accelerate the cell kinetics dilution.

Glyn: There are clinical procedures which can culture or these cells from peripheral blood a process called “mobilization”. These use growth factors to stimulate proliferation of the small stem cell numbers in peripheral blood.

What is the morphology of stem cells? 

James: Since tissue stem cells are difficult to identify with certainty, exact morphological distinctions have not been assigned to them as a group.  However, there is a widely accepted concept that they are smaller than other cells in their tissue of residence, because size fractionation can often enrich for stem cell transplantation activity.  However, this basis for discrimination is not sufficient for accurate and precise identification and quantification.

How can we know which type of stem cell can be cultured and expansion? 

James: If a culture of human tissue cells exhibits growth cessation with serial passage, without significant cell death (though it may occur dramatically at the end of a culture period due to apoptosis of terminally differentiated cells), then the responsible stem cells divide with too high a frequency of asymmetric self-renewal to be expanded. The only reported human tissue cultures that do not show growth cessation with serial passage are cells from individuals with p53 gene mutations. P53 has been shown to be required for asymmetric self-renewal in tissue stem cells.

How can we know which type of stem cell can be cultured and expansion? 

James: If a culture of human tissue cells exhibits growth cessation with serial passage, without significant cell death (though it may occur dramatically at the end of a culture period due to apoptosis of terminally differentiated cells), then the responsible stem cells divide with too high a frequency of asymmetric self-renewal to be expanded.  The only reported human tissue cultures that do not show growth cessation with serial passage are cells from individuals with p53 gene mutations.  P53 has been shown to be required for asymmetric self-renewal in tissue stem cells.

What are mammary stem cells? 

James: Tissue stem cells responsible for the renewal of mammary gland tissues.

What are cancer stem cells? 

James: Cells found in human tumors that have the ability to propagate the tumor cells in animal xenografts.  Often, though not always, the propagating cells are a small fraction of total tumor cells.

Why is it important to characterize stem cells when they are intended for clinical applications?

James: As noted early in the panel discussion, knowing the dose and quality of the stem cells in treatment preparations is essential for good clinical trial design, interpreting treatment outcomes, and replicating clinical findings.

Glyn: In bone marrow transplant of hematopoietic stem cells adequate levels of CD34+ cells are known to be associated with successful engraftment of donor bone marrow.

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