Stem Cells

Stem cells are unique in their capacity to regenerate the different cell types in the body.  Such regenerated cells have potential use in the treatment of various different diseases, including Parkinson's, Alzheimer's, diabetes and possibly even complete organ failure.  Before these stems cells can be used in regenerative medicine, sufficient quantities must be produced for research and production purposes.  One of the greatest limitations to stem cell technology at this time is the inadequate supply of quality, pluripotent stem cells.  Culture of stem cells is very time and resource consuming and often the cells will undergo premature differentiation.  If we as biochemical engineers can develop technologies that will allow us to generate large quantities of stem cells while inhibiting differentiation, the future applications of this technology for biomedicine will be greatly enhanced.    Thus, we are undertaking a collaborative project with scientists at the Kennedy Krieger Institute to develop methods for culturing large populations of pluripotent stem cells.

Stem Cell Culture


  1. hES cell were grown on feeder layers of mouse embryonic fibroblasts (MEF)
  2. Growth medium contained serum from calf or bovine sources
  3. Cells are not suitable for use in humans
    1. Risk of animal pathogens
    2. Possible immune rejection


  1. No feeder layer
  2. Grown on Matrigel (murine origin) or other growth matrix
  3. MEF conditioned growth media
  4. Added growth factors may be important
    1. leukemia inhibitory factor (LIF)
    2. basic fibroblast growth factor (bFGF)
    3. stem cell factor (SCF)

Ideal hES Cell Growth

  1. No feeder layer
  2. Growth on a simple growth matrix (Collagen?)
  3. Completely chemically defined media
  4. No contact with animal cells or proteins
    1. NO risk of animal pathogens
    2. Less possibility of immune rejection

Current Projects

With the overall goal of improving stem cell culture methods and moving toward the ideal hES cell growth conditions, we are currently involved with several projects:

1. Identification of hES cell growth factors
Certain cell lines make better feeder layers than others for hES cell growth.  In an attempt to understand this phenomenon, we are using DNA microarray technology in order to investigate the proteins transcribed by several different feeder layer cell lines.  We will compare the overall gene transcription and will use this data to identify factors that help maintain stem cell growth and pluripotency.

hES cells stained with Oct-4, a marker for pluripotency

2. Stem Cell differentiation using proteins
Studies have shown that endosomal release is a major limitation for delivery of cargo proteins to the cytosol via cell penetrating peptides.  The release of cell penetrating peptide linked fluorescent proteins was examined to determine the degree of restriction of transport from the media entirely through to the cytosol in multiple cell lines including HEK293, N18-RE-105, Hippocampal slices, and Lonza human neural progenitor cells.  However, proteins were still primarily endosomally localized.  Photochemical internalization is examined as a means to increase cytosolic delivery of increased amounts of natured protein and shown to significantly change the distribution of cellular fluorescence.  Finally, transcription factors previously shown to regulate dopaminergic neuronal development and differentiation were expressed as TAT-fusion proteins and were shown to be internalized by protein transduction.  The results show the groundwork for stem cell differentiation using proteins in place of DNA transfection.

The goal is to develop a strategy to enhance the neuronal differentiation through the introduction of transcription factors known to be vital to normal development of the nervous system.  An alternative strategy to viral vector delivery of desired genes is the direct delivery of purified proteins.  Desired transcription factors are expressed using mammalian transient expression.  The proteins are shown to be functionally active by EMSA and nuclear localization.  Though primarily endosomally localized, TAMRA labeling and confocal microscopy show that the transcription factors are preferentially transported to the nucleus when released from endosomes after extracellular incubation with living cells.  These purified proteins of interest can be used to drive neuronal protein expression in neuroblastoma and commercially obtained neural progenitor cells in a manner similar to transfection with the gene

3. Identification of endocrine progenitor cells in adult human pancreas
Diabetes mellitus (DM) is a chronic metabolic disorder caused by an absolute or relative deficiency of insulin, an anabolic hormone. Insulin is produced by the beta cells of the islets of Langerhans located in the pancreas, and the absence, destruction, or other loss of these cells results in type 1 Diabetes (insulin-dependent diabetes mellitus [IDDM]).  The monocellular nature of this disease and endocrine action of insulin make this disease an excellent candidate for cellular therapy. The promise of a cellular therapy for type 1 diabetes has been sparked by the introduction of novel immunosuppressive regimens used in conjunction with cadaveric islet transplantation.

Acinar-to-ductal metaplasia (ADM) can lead to islet neogenesis in both human and rodent. The goal of the study is to identify a source of endocrine progenitor cells in cultured adult human pancreatic tissue undergoing ADM.  The hypothesis is that the processes of harvesting and culture of cadaveric islets for transplantation trigger ADM and results in the generation of NGN3+/CD133+ endocrine progenitor cells.

Tissue prepared for islet transplantation is cultured for four days. During this period, changes in expression of amylase, cytokeratin 19, Mist1, Sox9 and Neurogenin 3 (NGN3) were measured by immunohistochemistry and RTPCR. Cells expressing the stem and progenitor cell marker CD133 were detected by immunohistochemistry and FACS, and isolated using immunomagnetic beads.

Results: CD133 is expressed on the luminal ductal surface of the human adult pancreas and by cells within clinical islet preparation tissue. Maintenance of islets under standard conditions prior to transplantation causes ADM. During this time, the percentage and total number of CD133+ and CD133+/NGN3+ cells increase.


© 2009 Johns Hopkins University