Engineering environments to prolong in vitro survival of human hematopoietic stem cells.
abnormalities in the bone marrow can have very severe consequences, which can be
observed in many bone marrow-related diseases, the most well known one being leukemia.
To date very limited treatment options exist for theses diseases and generally a stem
cell transplant is required for a full cure. These transplants are not only risky but
often fail at the stage of search for ... read morea compatible donor. A major reason for the absence
of potent treatment alternatives is that many molecular and cellular interactions within
the bone marrow are still unknown. To advance the cure of bone marrow-related diseases,
several groups are trying to develop a sustainable and functional human bone marrow
model that can serve two long-term purposes: * Assess the effects of new drugs on bone
marrow cells prior to human trials. This will not only remove xenogeneic risks, but
likely also reduce the need for animal testing. * Provide long-term proliferation of
whole bone marrow in vitro as a practical and cost effective source of stem cells. These
cells can be used for research but also help to establish a bank with a wide range of
stem cells that will facilitate donor matching for transplants. We have worked towards
achieving these long-term goals by designing environments that extend hematopoietic stem
cell (HSC) culture time in vitro. Specifically we increased HSC survival rate by: *
Determining an optimal medium for stem cell engraftment that supports both proliferation
and quiescence. * Adding adipocytes to currently established feeder layer models to
inhibit stem cell differentiation. * Creating a 3D construct and perfusion system that
supports long-term culture. Using our methods we concluded the following: * There are
two CD34+ populations within the bone marrow that differ in size. * The smaller cells
are more quiescent and possibly more primitive than the larger cells. * hMSCs
differentiation potential rapidly declines when expanding using traditional culture
methods. * The use of the conventional markers for hMSCs does not suffice in the
definition of a true stem cell. * The use of the more novel markers CD146 and CD271
would more likely be appropriate markers of stemness. * hASCs had an increased CD34
expression when compared to hMSCs. * An improvement of CD34+ in vitro survival rate was
achieved when using a feeder layer containing adipocytes. * Direct cell-cell contact is
necessary for the expansion of the hHSCs. * Long-term culture 2D is limited by the
functionality or peeling of the feeder layers. Therefore a construct for 3D bone marrow
culture is of great necessity. * Using a multiple seeding method one can obtain a
confluent 3D structure on a silk scaffold. * A unidirectional slow perfusion bioreactor
is a promising device to maintain in vitro 3D bone marrow
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Biomedical Engineering.
Advisor: David Kaplan.
Committee: Catherine Kuo, Pamela Yelick, and Clifford Rosen.
Keyword: Biomedical engineering.read less