Dr. Thomas is the Vice President of Research & Development of CardioVascular BioTherapeutics. He is a very well respected scientist in stem cell research, and he has been credited with the discovery of the fibroblast growth factor (FGF-1). He presented a fascinating discussion today regarding the past, present, and future developments of stem cell research.Stem cells have a few basic characteristics. They are, by definition, self-renewing, dedifferentiated, and they can differentiate into ectodermal, mesodermal, and/or endodermal cells. Stem cells are self-renewed because after cell division, a number of the daughter cells remain as ‘uncommitted daughter stem cells’. There are three basic levels of differentiation that stem cells are capable of: totipotency, pluriotency, and mulipotency. Totipotency is not discussed in Dr. Thomas’s discussion, but he does make a significant distinction about the differences between plutipotent stem cells and multipotent stem cells. In basic terms, embryonic stem cells (isolated from a blastocyst) are pluripotent and have the capability to differentiate into a broad range of cells. Multipotent cells, although still very useful in regenerative medicine, have a more limited range of cells that they can differentiate into.
To maintain undifferentiated “stem” state cells, cultured embryonic stem cells from mice require leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP-4). Interestingly, human do not require either of those molecules, but human embryonic stem cells instead require fibroblast growth factor (FGF). There are 4 FGF receptors, and each contain Ig-like domains. The FGFs bind between domains 2 and 3. Three of the receptors come in an alternate sequence, and this impacts the ability of different FGF receptors to distinguish between them. FGF-1 is unique in that it potently activates all 7 FGFR isoforms.
FGF maintains the dedifferentiated state of human embryonic stem cells. Dr. Thomas provided a great example where stem cells were cultured in a fibroblast feeder layer of 4 ng/mL FGF-2, a fibroblast conditioned medium containing 8ng/mL FGF-2, and a dish containing only 100 ng/mL FGF-2. The high levels of FGF-2 support growth of embryonic stem cells in the absence of fibroblast conditioned medium. Human embryonic stem cells generate their own support cells. In response to FGF stimulation, support cells express and secrete IGF-II and TGFβ, thus FGF is a trigger that induced support of human embryonic stem cells. IGF-II supports proliferation TGFβ maintains the dedifferentiation of embryonic stem cells.
Is it possible for somatic cells to be reprogrammed to become pluripotent undifferentiated cells? YES! If dermal fibroblasts are transfected with genes for 4 transcription factors (Oct4, Sox2, Klf4, c-Myc or Oct4, Sox2, Nanog, Lin28), they will become induced pluripotent stem (iPS) cells. These cells are virtually identical to embryonic stem cells with respect to morphology, growth factors needed to maintain dedifferentiated “stem” state, epigenetic status, gene expression profile, and they will differentiate on withdrawal of the growth factors previously mentioned. Oct4 & Sox2 are master transcription factors that bind adjacent DNA sequences. There are approximately 400 gene promoters that bind Oct4/Sox2. These transcription factors increase expression of genes that enhance dedifferentiation and inhibit those that promote differentiation. The functions of Nanog/Lin28 and Klf-4/c-myc are not as well understood. Mouse and human iPS cells from dermal fibroblasts without Myc retroviral transfection result in a lower frequency but higher specificity than Myc transfection. These induced pluripotent cells have identical gene expression profiles with embryonic stem cells. Lastly, it is important to note that no tumors have been detected in iPS cells without Myc whereas those co-transfected with Myc do show about a 15% cumulative incidence rate of tumors in mice at four months of age.
Hematopoetic stem cells (HSCs) are adult stem cells. Bone marrow HSCs proliferate and differentiate into all of the major circulating blood cells. HSCs are capable of differentiating into a number of myeloid and lympoid cells, such as: granulocytes, macrophages, neutrophils, basophils, eosinophils, mast cells, red blood cells, platelets, natural killer cells, B cells, and T cells. HSCs express the receptors FGFR1, R3, and R4 and they bind FGF1. In the mouse model, embryonic stem cells missing FGFR1 exhibit defective hematopoietic development. Interestingly, FGF-1 supports growth of hematopoietic stem cells in serum-free culture. Implantation into radiation-compromised mice of hematopoietic stem cells expanded by FGF-1 led to long-term survival and full repopulations (i.e. myeloid cells, lymphoid cells, erythroid cells).
FGF induces reversible increases of leukocytes in clinical trials. FGF-2 delivered i.v. at a concentration of greater than 24μg/kg resulted in transiently increased leukocyte counts in approximately 50% of patients in a coronary artery disease trial. FGF-2 i.v. infusion at a concentration of 75-150 μg/kg in the stroke trial resulted in about a 2 fold increase in leukocyte counts. These results occurred within 2 days of treatment and they are not associated with fever, infection, or other adverse effects.
FGF also plays a role in neurogenesis. Studies performed in rodents have shown that neural stem cells reside in the subgranular zone of the hippocampal dentate gyrus and the subventricular zone of the olfactory bulb. It has been shown that FGF drives neurogenesis. When FGF-2 is added at a concentration of 10ng/mL to a culture of neural stem/progenitor cells, the cells differentiated into neurons. Another study was done in perinatal rats (1-3 weeks) and adolescent rats (1 month). FGF was administered at a dose of 5 ng/gm of body weight by a single intracerebroventricular or subcutaneous injection. From 6 to 8 hours after FGF was administered, a mitotic pulse transiently labeled the cells that are dividing and showed a 2 to 3-fold increase in newly dividing subventricular zone neurons. It is also important to note that FGF-2 and –1 can cross the blood brain barrier.
It has been shown that FGF can control the differentiation of embryonic stem cells into cardiomyocytes. High FGF-2 concentration (i.e. 20 ng/mL) will inhibit embryonic stem cell differentiation into cardiomyocytes because it down regulates the FGFR1 and FGFR2 genes. Low FGF-2 concentration (1 ng/mL) promotes embryonic stem cell differentiation into cardiomyocytes and upregulates the FGFR3 and FGFR4 genes. Anti-FGF antibodies inhibit a low spontaneous rate of cardiomyocyte differentiation from embryonic stem cells. FGF-2 dose response of cardiomyocyte differentiation appears to follow a typical agonist bell-shaped curve.
Differentiation of embryonic stem cells into cardiomyocytes requires FGFR1. When the FGFR1 gene is knocked out in the mouse model, it results in fewer beating foci, a slow beating rate (i.e. 47 versus 99 beats/min), little expression of myocardial genes, and the expression of markers for other mesodermal lineages.
Dr. Thomas discussed a few characteristics of resident cardiac stem/progenitor cells. These results came from studies of neonatal mouse heart cells in culture. Cardiac stem/progenitors cells are less than 1% of all cultured heart cells, and approximately 5% spontaneously differentiate into cardiomyocytes that can contract (synchronous beating). FGF-2 can promote differentiation into cardiomyocytes. Interestingly, when the FGF-2 gene is complemented back into FGF-2 (-/-) gene knockout mice, a lack of differentiation is observed by cardiac stem cells.
Adult cardiogenesis, a reversal of terminal differentiation, has also been shown to occur in adult rats. Out of the 45 growth factors tested in this study, FGF-1 was the most active. It was concluded that FGFs can transiently reverse the differentiation program.
Dr. Thomas listed a number of FGF responsive stem cell systems: embryonic stem cells, hematopoietic stem cells, neural stem cells, cardiac stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, pancreatic stem cells, intestinal stem cells, epidermal stem cells, hair follicle stem cells, and germ stem cells. In conclusion, one of the most important questions to as is: what potential implications does FGF therapy have? In stem cells, FGF can be used to maintain, expand, or contribute to the differentiation of stem/progenitor cells. In addition, angiogenic activity may provide nutrients to support stem cell niches. In cells that have already been differentiated, FGF drives replication “terminally differentiated” cells (i.e. cardiomyocytes) and will also drive the replication of primary cells under stress, such as ischemia/hypoxia and tissue damage.
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