Human Adipose-derived Multilineage Precursor Cells: Science, Ethics and Implications for Cardiac Cell Therapy Allison Chamberlain

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1 Human Adipose-derived Multilineage Precursor Cells: Science, Ethics and Implications for Cardiac Cell Therapy Allison Chamberlain One of the most promising sectors of biomedical research deals with stem cells. Stem cells, or cells that have yet to differentiate into a specific cell lineage, are found in many bodily tissues and are capable of undergoing differentiation into one or more specialized cell types. Being able to harness these differentiation capabilities is highly anticipated in medicine; the clinical implications stem cells have on cell therapy and tissue regeneration are incredible. Stem cells could be turned into brain cells to cure degenerative diseases like Parkinson s disease or nerve cells to restore feeling in paralysis patients. These cells could also regenerate bone for patients with osteoporosis or cartilage to repair older joints. In looking forward to these future applications, scientists and doctors are working hard to get this translational research from the bench to the bedside as quickly as possible. 1 Two main categories of stem cells exist: embryo-derived stem cells (ES cells) and adultderived stem cells. ES cells have been shown to have the greatest differentiation plasticity due to their immaturity, but because they are derived from human blastocysts, a type of nascent human life, many people are opposed to research with these cells. Additionally, due to their pluripotency, ES cells have a tendency to form multi-lineage tumors called teratomas after injection into animal models. i 2,3 Due to both the ethical controversy over the manipulation of human embryos and the threat of causing cancerous tumors, scientists turned more focus onto adult-derived stem cells. Found in various tissues within the adult body, these cells also have regenerative capabilities, but because they are further along in the differentiation process than ES cells, they are not as pluripotent. Within the last decade however, researchers have found adult stem cells to have far greater differentiation potentials than originally thought, thus bolstering more research efforts. Stem cells derived from bone marrow have been studied more than any other adultderived stem cell type. The regenerative capacities of hematopoetic stem cells in marrow have been understood for years; these are the cells responsible for replenishing the body s supply of red blood cells. Through research with these cells, researchers discovered a second population of stem cells, termed mesenchymal stem cells that also have regenerative capabilities. ii Studies with this type of stem cell have broken the traditional assumption that adult-derived stem cells were fated to only ever become the tissue type in which they reside. These mesenchymal bone marrow-derived stem cells have been successfully differentiated into cartilage, muscle and bone cells. 4-7 This mesenchymal stem cell source is promising, but extracting bone marrow is a very invasive procedure. Requiring either drilling into major bones or administering a powerful stem i Pluripotency refers to having great developmental plasticity; ES cells can produce all cell types. ii Mesenchymal refers to the capability to create connective tissues, blood, lymphatics, bone and cartilage. 1

2 cell mobilization drug called G-CSF, such rigorous harvesting procedures can be very harmful to patients who are already afflicted with debilitating degenerative diseases. Scientists therefore began examining other adult tissue types for stem cell sources and found a population of stromal cells within fat that had mesenchymal differentiation properties similar to the stem cells in bone marrow. Termed human adipose-derived multilineage precursor (HAMP) cells, these progenitor cells have successfully been differentiated in vitro into adipogeneic (fat) 8, chondrogenic (cartilage) 9, myogenic (muscle) 10,11, osteogenic (bone) 8, and neuronal (nerve) cells. 12,13 What makes these HAMP cells more appealing scientifically is their abundance, ease of extraction and sustainability in culture. In comparison to bone marrow-derived cells, HAMP cells are available in much greater quantities considering the fact that nearly 40% of Americans are overweight. They are easily obtained through liposuction or abdominoplasty procedures done routinely in plastic surgery operating rooms, yielding upwards of 2 million cells per abdominoplasty section. Contrastingly, stem cells within marrow are present in amounts of approximately 1 in 100,000 nucleated cells, and there is only a very small volume of marrow that can be collected with limited morbidity. 14 HAMP cells also require only about 5% of the cell number used for marrow cells to reach initial confluence in one week. Suggestive of an increased proliferative potential, this could indicate generation of a clinically effective dose more rapidly than for an equivalent number of marrow-derived cells. 14 Of the potential uses for HAMP cells, the regeneration of cardiomyocytes for cardiac repair is one of the most important. Since cardiovascular disease kills more Americans than any other cause, there is a compelling reason to research treatments and procedures that would help alleviate this crisis. In 2001 alone, there were 502,189 heart attack deaths in the US, and 13.2 million people living with forms of coronary heart disease. iii Research using adipo-derived stem cells to improve the function and stability of hearts damaged by heart attacks could not be more valuable. The American Heart Association realizes the potential benefits of stem cell research, calling it the most promising medical and scientific research to help fight cardiovascular disease. iv They fund many human stem cell research projects, but only ones that involve adult stem cells. The AHA acknowledges the greater pluripotency of embryonic stem cells, but does not directly support research involving embryonic or fetal tissue. Again, such a stance illustrates the pervasiveness of the ethical controversy over ES cells. If research with HAMP cells indicates that these cells can help damaged hearts, there is little doubt that the AHA and every hospital will support it fully. In order to eventually restore heart tissue in damaged hearts, HAMP cells must be induced down the cardiomyocyte pathway. However, cardiomyocytes, which are mature heart cells, are unique muscle cells which have characteristics unlike any other type of skeletal or smooth muscle cell. Among these is the ability to beat, so a primary experiment in determining if HAMP cells have cardiomyogenic potential is to see if they can be induced to contract. Based iii Statistics from AMAonline. iv From AMAonline. 2

3 upon previous success in turning these cells into other cell types, they should be able to differentiate down the cardiomyogenic lineage in vitro upon addition of the correct growth factors. In replicating the protocol described by Rangappa, et al, HAMP cells were exposed to the DNA methyltransferase inhibitor 5-azacytidine for 24 hours in culture, and then monitored by microscope for one month for cardiomyogenic characteristics like binucleation, extended cytoplasmic processes, ball-like formation, spontaneous beating and expression of cardiac specific markers like Nkx2.5, Troponin I, and connexin-43. v 15 Some cells did demonstrate binucleation and cytoplasmic processes, but there was never any ball-like formation, spontaneous beating or expression of cardiac-specific markers. This lack of cardiomyogenic differentiation may have been due to inter-species differences; the 5-azacytidine concentrations and cell densities may have needed alterations to account for any differences between adipoderived stem cells from rabbits and those derived from humans. Such in vitro work with these cells allows for more controlled differentiation studies, but equally valuable is how these cells react in vivo. When injected into damaged hearts, stem cells are in the most differentiation-conducive environment possible. Damaged tissues release a milieu of induction and repair factors that are meant to attract bodily stem cells specifically for repair. This precise microenvironment is nearly impossible to replicate in vitro, so in vivo injection experiments should accompany any work in culture. To assess the behavior of HAMP cells in live animal models, HAMP cells labeled with a fluorescent marker, BrdU and iron were injected into both healthy mouse hearts and infarcted mouse hearts. vi Our studies revealed that in both infarcted and non-infarcted hearts, the HAMP cells remained in the hearts near the injection sites in the left ventricular walls. MRI data on the infarcted animals revealed that the cells engrafted into the heart walls and stayed in and around the damaged left ventricle for at least 28 days after injection. In comparison to historical control mice with infarcts of similar size, functional analysis of both left ventricular end systolic volumes and left ventricular ejection fractions revealed trends towards functional improvements in the hearts injected with HAMP cells. vii Once the mice were euthanized, their hearts were paraffin-embedded and sectioned to determine the precise locations and morphologies of the cells remaining in the heart tissue. Using both anti-brdu immunohistochemistry and the fluorescent signal from the DiI labels, the HAMP cells were located within the left ventricular walls. Stained sections revealed that the cells looked more like elongated fibroblasts than healthy rounded cardiomyocytes. Such morphological analysis suggested that the HAMP cells did not actually turn into cardiomyocytes. v 5-azacytidine is a genetic inhibitor that has been shown to induce cardiomyogenic differentiation in culture. vi BrdU is a thymine analog that incorporates itself into the DNA of cells and is used as a marker that can later be visualized through immunohistochemistry. The fluorescent label was DiI. Infarcted means damaged after heart attack. vii Comparisons had to be made to historical controls because the two control mice in this experiment died due to infarcts that were too large. 3

4 However, possibly by engraftment alone, they were able to demonstrate stabilizing trends by providing some type of structural stability to the weakened left ventricular walls. When the sections were tested for Nkx2.5, Troponin I and connexin-43, the only possible positive results came from Troponin I, indicating either that the HAMP cells differentiated to a point and then stopped, or that a technical error, like non-specific binding between the Troponin I antibody and the HAMP cells, was causing a false signal. More in vivo injection studies with different cellular markers, like more precise genetic tags, could confirm or deny HAMP differentiation. The results of this HAMP cell research indicating trends toward functional improvement without actual cardiomyogenic differentiation are congruent with findings in similar cardiac adult-stem cell studies. Experiments with injections of hematopoetic stem cells into infarcted hearts have not resulted in cardiomyogenic differentiation either, but slight improvements in function have been reported Such inconclusive results make the Food and Drug Administration hesitant to approve human trials in the US right now, even when other countries like France and Germany have been injecting human heart with various types of stem cells since Even though cardiac cell therapy research is highly anticipated, it must not be rushed. Scientists need to do more research to assure that the injected cells will not differentiate into undesired cell types like bone or cause potentially fatal conditions like arrhythmia. viii Not only will future therapies eventually help millions recover from heart attacks, but fame and financial incentives are expected as well. In this regard, it is frustrating that other countries may be ahead of the US in terms of human trials, but America needs to keep its ethical and safety standards high so that no tragic accidents occur. The FDA s reluctance is appropriate for the time being; much research remains to be done in animals before cardiac cell therapy research with HAMP cells, or any type of stem cell, moves into the human trial stage. Stem cell research with HAMP cells is extremely promising however. As more induction factors are identified and understood, scientists can begin to direct these cells down certain lineages in culture before implanting them as precursor brain cells or cardiomyocytes. More research can even determine if these cells can home to injured areas so that direct organ injections are unnecessary. Because of their abundance, ease of acquisition, ethical acceptability, and comparable differentiation capabilities, HAMP cells are a favorable stem cell source to research for future cell therapy applications. References: 1. Chien, K. Lost in Translation. Nature (2004). 2. Wakitani, S. et al. Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 42, (2003). viii Arrhythmia is a heart condition resulting from unsynchronized beating. This can occur if all cells in the heart are not beating simultaneously, and it can be fatal. 4

5 3. Brickman, J. M. & Burdon, T. G. Pluripotency and tumorigenicity. Nat Genet 32, (2002). 4. Mackay, A. et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4, (1998). 5. Fukuda, K. Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes. C R Biol 325, (2002). 6. Makino, S. et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103, (1999). 7. Banfi, A. et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Experimental Hematology 28, (2000). 8. Zuk, P. A. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7, (2001). 9. Awad, H. A., Wickham, M. Q., Leddy, H. A., Gimble, J. M. & Guilak, F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials 25, (2004). 10. Mizuno, H. [Versatility of adipose tissue as a source of stem cells]. J Nippon Med Sch 70, (2003). 11. Wickham, M. Q., Erickson, G. R., Gimble, J. M., Vail, T. P. & Guilak, F. Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin Orthop, (2003). 12. Safford, K. M. et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 294, (2002). 13. Ashjian, P. H. et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg 111, (2003). 14. Fraser, J. K., Schreiber, R. E., Zuk, P. A. & Hedrick, M. H. Adult stem cell therapy for the heart. The International Journal of Biochemistry and Cell Biology (2004). 15. Rangappa, S., Fen, C., Lee, E. H., Bongso, A. & Kwang, E. S. Transformation of Adult Mesenchymal Stem Cells Isolated From the Fatty Tissue Into Cardiomyocytes. Annals of Thoracic Surgery 75, (2003). 16. Balsam, L. B. et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature (2004). 17. Murry, C. E. et al. Haematopoietic stem cells do not transdifferentiate in myocardial infarcts. Nature (2004). 18. Shake, J. G. et al. Mesenchymal stem cell implantation in a swine myocardial infarct model. Annals of Thoracic Surgery 73, (2002). 19. Couzin, J. & Vogel, G. Renovating the Heart. Science 304, (2004). 5