Ronquillo 1 Seth H Ronquillo MCD BIO 98T March 18, 2011 Final Project The Birth of an Alternative: Using Umbilical Cord Stem Cells to Improve Stem Cell Research Stem cells are intriguing. They are so dynamic, so puzzling, and they contain many of biology’s deepest mysteries. Generally, cellular stemness is characterized by a cell’s self-renewal and potency, the ability to differentiate into specific cell types. In reality though, the science of stem cells goes beyond these distinctions. From studying human development to creating new therapeutic alternatives for diseases, embryonic stem cell (ESC) research promises numerous benefits as it has progressed extensively in the past decade. But because of the ethical issues surrounding the destruction of human embryos, other alternatives have had to be found. So far, adult stem cells (ASCs) have been used for therapeutic purposes such as bone marrow transplants. However, donor DNA-matching has been an enduring problem with this approach. A few years ago, Shinya Yamanaka discovered induced pluripotent stem cells (iPSCs), which derives pluripotent stem cells from somatic cells, thereby overcoming both ethical and donormatching issues. Although iPSC presents much promise, the newness of this technology makes it inadequate for immediate use. Now, newer discoveries have been made. Aside from ESCs, ASCs, and iPSCs, stem cells have also been found in postnatal tissue, particularly in the umbilical cord. These umbilical cordderived stem cells have been proposed to treat illnesses ranging from Parkinsonism to leukemia in both animal models and humans alike. Above all, these stem cells face less ethical and

Ronquillo 2 technical concerns than the other three methods, when it comes to research utility. Even though uncovering the secrets behind umbilical cord-derived stem cells is still in the works, this research deserves much attention as it offer beneficial applications without the overwhelming dilemma. Before the umbilical cord can be understood, one has to look first at the consecutive events during human development that lead to cord formation (Fig. 1). When the egg and sperm combine during fertilization, they form a single-celled, totipotent zygote, capable of becoming any cell type. Before it becomes a full-pledged embryo though, this zygote divides to become a colony of cells, eventually becoming the two-layered blastocyst. Comprised of the external trophoblast, which would form the embryo’s supportive tissue, and the inner cell mass (ICM),

Figure 1 – The sperm fertilizes the egg to form the zygote which divides to later become the two-layered blastocyst (Days 1 – 4). After implantation (Day 7), the embryo develops by differentiating into the three germ layers (Day 12). It also starts to recede from the trophoblast and the placenta, but the allantois connects the two together with a connecting stalk (Day 18). Eventually, the connecting stalk brings about the umbilical cord (Day23). (Image Source: “Human Embryogenesis.” Wikipedia. Wikimedia Commons. September, 2010.)

Ronquillo 3 which would become the embryo itself, 1 this blastocyst is the first stage of cellular differentiation from totipotency to pluripotency. Days after fertilization, the still-dividing embryo has to implant into its mother’s uterus. “If implantation succeeds, the structures that support the embryo begin to form.”1 Interestingly enough, the trophoblast and ICM work hand-in-hand from this stage onward to ensure a healthy embryo. As development progresses, the mesoderm, one of the germ layers created by the ICM, extends from the posterior end of the embryo and combines with the trophoblast to form the extraembryonic placenta.2 This mesodermal outgrowth also gives rise to the allantois, a primitive component of the umbilical cord (Fig. 2).2 Because the mesoderm is responsible for the creation of blood and related tissues in the developing embryo,1
Figure 2 – After implantation, the mesoderm creates an outgrowth that extends from the posterior end of the embryo. This outgrowth forms the allantois, an early constituent of the umbilical cord, and combines with the trophoblast to form the placenta.2

the allantois in itself has hematopoietic potential and, in time, brings about blood vessels.2 From hereon, the embryo starts to recede from the placenta, but the two are still attached together by a connecting stalk formed by the allantois and its blood vessels.3 Within 10 weeks, this connecting stalk develops into a recognizable umbilical cord which functions as a blood channel between the fetus and the placenta3 for waste and nutrient exchange.2 As the fetus’s connection to the outside world, the importance of the umbilical cord is obvious. More importantly, the significance of the umbilical cord to scientists today lies in its potential as a source of stem cells for research. Historically, stem cells in the umbilical cord, particularly in the umbilical cord blood (UCB), have been commonly used as a source of hematopoietic stem cells (HSCs).4 In the

Ronquillo 4 hematopoietic (i.e. blood) cell hierarchy, HSCs, recognizable by the protein CD34, are the stem cells that give rise to progenitor cells. These progenitors expand and ultimately bring about the specific types of fully differentiated blood cells in the body, including the oxygen-carrying red blood cell and the disease-fighting white blood cell. In one experiment, researchers sought to identify the hematopoietic progenitor cells within the umbilical cord that give rise into the three types of blood cells: endothelial, myeloid, and lymphoid. Respectively, these cell types give rise to blood vessels, platelets and red blood cells, and white blood cells.5 Knowing that HSCs as adult stem cells express the protein CD34 and that progenitors express CD133, Ramos et al hypothesized that they could isolate the hematopoietic progenitors of the UCB by using these two markers. After isolating the putative progenitor cells, the researchers wanted to determine the cell’s differentiation potential into the endothelial, myeloid, and lymphoid lineages, but first, they allowed the cell population to expand and proliferate. As the cell colony formed and grew, Ramos et al found progenitor cells and continued to differentiate the progenitors into the cell types of interest. Once the researchers recognized that certain percentages of the colony exhibited characteristics of the desired cell types, they divided up the cells based on their characteristics (Fig. 3). They noticed morphological distinctions as well as
Figure 3 – Since the cells are able to self-renew, about 50 percent of the colony remained multipotent. 34 percent had endothelial markers, 21 percent had myeloid markers, and 44 percent had lymphoid markers. (E – endothelial; M – myeloid; L – lymphoid; B/T – types of lymphoid cells).5

specific gene expression markers amongst the cell groups, indicating successful differentiation.5

Ronquillo 5 To test the full utility of the hematopoietic cells in vivo, researchers transplanted the cells into mice with limited blood flow in their legs (Fig. 4). At the beginning of the transplantation experiment, Ramos et al cut the blood vessels in one of the legs of the mouse subjects. As a result, no blood flow is observable in this region of the mice’s body.
Figure 4 – After detaching the blood vessels on the hind leg of a mouse, researchers transplanted endothelial cells in the affected region (rightmost column). 28 days later, noticeable blood flow can be seen on the recipient mouse’s leg, in comparison to the other mice that did not get the transplant (left and middle columns). (Blue – no blood flow/no fluorescence; Red – high blood flow).5

Subsequently, the researchers injected differentiated endothelial (i.e. blood vessel) cells into the

affected area. After much inspection, they saw the reemergence of blood flow on the transplantrecipient mice’s leg, unlike the control group that did not receive the transplantation. These results show that revascularization, due to the injected cells, improved the blood flow in the mice’s leg.5 While this study proves that a hematopoietic progenitor exists within the UCB and that different, functional hematopoietic cell types are derivable from the multipotent UCB-HSCs, others show that UCB has a greater potential beyond HSCs. Besides HSCs, four other progenitor and stem cell types have been discovered in UCB: 1) Multipotent progenitor cells (MPSCs) are highly proliferative and can differentiate into osteoblasts, myoblasts, and hepatocytes. 2)

Ronquillo 6 Mesenchymal stem cells (MSCs), which are more commonly found in the bone marrow, are able to differentiate into adipocytes, neurons, as well as osteoblasts. 3) Rare unrestricted somatic stem cells (USSCs) have the ability to proliferate extensively without spontaneously differentiating; these cells have relatively longer telomeres and would allow researchers to have an efficient, genetically diverse source of cells, especially when it comes to stem cell research. In addition, USSCs can still be chemically coaxed into differentiating into the cell types found in the three germ layers. 4) Lastly, cord-blood derived embryonic-like stem cells (CBEs), as the name indicates, display some ESC characteristics, including the expression of Oct-4, but are not pluripotent. Nonetheless, they are capable of differentiating into neuronal, hepatic, skeletal, and blood vessel cells. Conclusively, scientists recognized that the genetic and morphological characteristics of these four cell groups are too different for them to be the same kinds of cells.4 Nevertheless, more study may still be required for these cells but, the multiplicity and the differences among them show the versatility of the stem cells in UCB. Another study has shown that certain cells lining the umbilical cord express the gene MUCIN1. These multipotent MUCIN1-expressing Cord Lining Epithelial Cells (CLEC-muc) are unique in that they are only found on the lining of the umbilical cord, and should therefore not be mistaken for other kinds of UCB stem cells. Profoundly groundbreaking, these cells are “novel” in that they carry both adult and embryonic stem cell characteristics. While they express gene markers found in epithelial stem cells, they are also highly proliferative, self-renewing, and express some of the most essential ESC genes: OCT-4, NANOG, SSEA-4, REX1, and SOX2. Additionally, these cells can differentiate into non-keratinized epithelia which comprise dermal tissue. In their experiment, Reza et al isolated CLEC-muc from a healthy umbilical cord. As the researchers put the cells into multiple passages, they noticed that CLEC-muc were continuously

Ronquillo 7 proliferative, indicating a characteristic of stem cells. One of the most interesting observations made was that these cells had gene markers identifiable with epithelial as well as mesenchymal stem cells. The researchers also used immunostaining and
Figure 5 – As indicated by the fluorescence, CLEC-muc expresses Oct-4, REX1, NANOG, SSEA-4, and SOX2. However, TERT is clearly not expressed. This indicates that CLEC-muc has a strong resemblance to ESCs, but the absence of TERT, among other characteristics like non-pluripotency, makes it different from ESCs.6

found that the cells expressed NANOG, OCT-4, REX1, SSEA-4, SOX2, but not TERT, indicating that CLEC-muc “may have some residual effect of ES cell-specific molecules” (Fig. 5).6 All in all, with its unusually hybrid properties, this novel line of cells can be used not just for therapeutic purposes but also to study the relationship between adult and embryonic stem cells. Certainly, stem cells in the umbilical cord may be only multipotent, but they come in so many different varieties that their collective multipotency is almost equivalent to an ESC’s pluripotency. From neurons to cardiomyocytes, scientists have been able to develop different cell types with pluripotent ESCs. However, as researchers focus more energy on ESC technology, it seems that they have forgotten the versatility of umbilical cord stem cells. Of particular noteworthiness are the MSCs found in the umbilical cord. Naturally, MSCs are derived from the mesoderm and can be found in an adult human bone marrow. However, from the umbilical cord-

Ronquillo 8 derived mesenchymal stem cells (hUC-MSCs), scientists were able to derive and isolate in vitro neural and other stem cells for therapeutic use. Already, they have tested these cells in rats with much positive results.7 In terms of neuronal applications, Weiss et al isolated umbilical cord matrix stem cells (UCMSCs), a genetic relative of hUC-MSCs. In vitro, they differentiated the multipotent cells and observed that the cells can actually become functional neurons. Originally, the acquired neuronal cells were characterized by nestin, a protein that is distinctive to undifferentiated neurons. However, after repeated passages, the cells expressed less nestin and more tyrosine hydroxylase (TH), a protein indicative of differentiated, working neurons. With these useful, differentiated neurons at hand, Weiss et al transplanted the cells into rats with Parkinson’s disease and looked for signs of recovery. Because Parkinsonism causes rats to rotate either only left-laterally or only right-laterally, the researchers used apomorphine-induced rotation studies, where they make rats go around in circles, to examine behavioral changes. Whereas rats that did not get the neuronal transplantation (the control group) were observed of constantly only being able to rotate on one side (either left or right), rats that were injected with the differentiated cells showed
Figure 6 – Researchers examined the number of dopaminergic, TH-neurons on the left and right sides of the brain of the rats. They found that there is an increase on the cell-count of TH cells in the brains of rats that were able to rotate on both directions. On the other hand, the control group, which did not get any transplant, had a constant number of TH cells. (White bar – transplant-recipient rats that were able to spin on both directions; Plaid bar – transplant-recipient rats that did not respond as much to treatment; Black bar – control group which had no transplantation).8

Ronquillo 9 major improvement; transplant-recipient rats demonstrated the ability to rotate on both left and right sides after multiple tests. Moreover, Weiss et al saw a significant increase in the number of well-functioning dopaminergic neurons in the brains of recipient rats in comparison to the control group (Fig. 6). Of equal importance is the fact that no brain tumors formed in the process.8 Aside from this Parkinsonian example, photoreceptor degeneration and type 1 diabetes have also been cured in rat models through MSCs from natal tissue, according to researchers. Specifically with the latter example, scientists observed insulin release and stable blood glucose levels in rat models with diabetes after undergoing an hUC-MSC transplant. Cardiomyocytes have also been successfully derived from hUC-MSC, and when transplanted into rats with cardiovascular problems, these hUC-MSC-derived cardiomyocytes improved the cardiac function of the rats.7 Ever so promising, umbilical cord-derived stem cells have indeed proven their ample therapeutic abilities in these experiments. The success of these studies is remarkable, but one has to understand that the research on umbilical cord stem cells is still young. Without a doubt, many questions are yet to be answered before these cells can be used to cure all human illnesses. Nevertheless, replacing bone marrow transplant (BMT) in treating blood illnesses like leukemia and anemia is presently the most common medicinal application of umbilical-cord derived stem cells. In Korea, umbilical cord blood is frozen after birth through cryopreservation for future use. These preserved cells can be later used for umbilical cord blood transplantation (UCBT) which has now replaced BMT in Korean hospitals when it comes to fighting blood-related diseases. Since sources of the grafts used in transplantation are often donors who are unrelated to the patients, treatment with UCBT can be initiated more readily than with BMT. Primarily, because they are immunologically naïve,

Ronquillo 10 stem cells from the umbilical cord can be used to timely treat illnesses without much concern for donor-patient matching.9 In one study conducted by Yoo et al in Korea, the effects of UCBT on sick children were recorded, and it turns out that complications are less likely to occur with UCBT than with BMT. Since a small number of stem cells reside in the umbilical cord and expanding them is not yet a possible option, the number of cells transplantable from natal tissue is limited and a single donor cannot suffice for the needs of a fully-grown, adult patient. To compensate for this deterrent, double-unit UCBT can be done on bigger (i.e. heavier, older) patients who needs more cells. This means that double dosages of UCB collected from different sources can be used without having to worry about injurious immunological reactions on the patient’s part.9 The many possible benefits of research on umbilical-cord derived stem cells are indeed tremendously exciting. From studying the chemical development of stem cell to applying the technology for therapeutic purposes, stem cells in the umbilical cord also pose, if any, minimal ethical obstacles for research. To begin with, unlike in ESC research, no embryos are destroyed in umbilical cord research. The many ethical and political issues that impede the success of ESC technology can thus be easily ignored. Manipulating ESCs can also be very inefficient and costly7, whereas stem cells in the umbilical cord are already partially differentiated and, hence, easier to work with.4 What’s more is that umbilical cord stem cells also possess some ESC characteristics, making them very multipurpose.6 Research on ASCs has also been generally more ethically favored than ESC research. By not having to use embryos, ASC research has garnered strong political support and has consequently undergone expanded efforts in the past few years. However, with old age, cells decrease their expansion and proliferative abilities, posing a drawback on the utility of aged

Ronquillo 11 ASCs. Equally worth mentioning is the fact that the differential capacities of older ASCs are significantly reduced, making them impractical in technical terms.7 Furthermore, stem cells can be found almost everywhere in postnatal tissue, from the placenta to the other extraembryonic tissues after birth.7 Adding onto its abundance, umbilical cord tissue is also often considered medical waste and is either discarded7 or frozen and stored for later use.4 Hence, there is no issue in its acquisition in comparison to the invasiveness of getting ASCs and the difficulty of getting new embryos for ESC research. This also means that the genetic diversity from the banks of frozen umbilical cord is greater than in the current sources of ESCs where the genes of upper-class whites are more predominantly represented. Umbilical-cord derived stem cells indeed give researchers the best of both worlds since they are the middle ground of embryonic and adult stem cells.6 Nonetheless, in spite of all the praise umbilical cord stem cells are receiving, it is a resource that is far from flawless. For one, the multipotency of umbilical cord stem cells is a double-edged sword. Because they are only multipotent, these stem cells can never be used to study the development of an embryo to a fetus. They may be used to study the relationship between embryonic and adult stem cells,6 but their limited potency posits a technical restriction for more in depth analyses. Among their other shortcomings, most of the therapeutic research on these cells, beyond redressing BMT, has only been done so far on animal models.7 They may have shown extraordinary results in these studies, but a human’s physiology is still different from a rat’s. More importantly, the current therapeutic application of umbilical-cord derived stem cells in UCBT is also still imperfect. 9 Stem cells in the umbilical cord may be immunologically naïve, but they are not immunologically innocent. Graft-versus-host disease (GvHD) happens when the immune system of the donated tissue reacts against the host tissue,

Ronquillo 12 creating a battle between the two tissues’ immunities—hence, the name graft-versus-host. This can cause serious complications on the patient’s skin and liver and is a pressing matter for umbilical cord stem cell research.10 Such side effects mar therapeutic procedures with umbilical cord stem cells in that GvHD takes over in 15 percent of patients after transplantation.9 While 15 percent may be a small amount, safety is always a top priority for researchers. Putting everything into perspective, from its benefits to its flaws, umbilical cord stem cell research is perhaps one of the most significant innovations in biology over the past years. Ironically though, it has been considerably overlooked as a promising resource. Doctors are restricted by the limitations on ASCs. Scientists struggle against the politics that impede them from making strides with ESCs. Researchers are boggled by the mystery surrounding the chemistry of iPSCs. Yet, in front of them are umbilical cord stem cells that lack the ethical and technical dilemma of these three methods combined. All that these umbilical cord stem cells need is the attention of researchers, so that they, as an amazing resource of cells, can live to the promise they embody.

Ronquillo 13 References 1. Scott, C. T. (2006). Stem Cell Now: From the Experiment That Shook the World to the New Politics of Life. New York: Pi Press, 2006. pgs 24-34 2. Dzierzak, E., et al. (2010). Placenta As a Source of Hematopoietic Stem Cells. Cell; August, 2010: pgs 361-366. 3. Kliman, H. J., (1998). The Umbilical Cord. Encyclopedia of Reproduction; September, 1998: http://www.med.yale.edu/obgyn/kliman/placenta/articles/EOR_UC/Umbilical_Cord.html. 4. Lee, M. W., et al (2010). Stem And Progenitor Cells in Human Umbilical Cord Blood. Int J Hermatol; June, 2010: pgs.45-49 5. Ramos, A. L., et al. (2010). Clonal Analysis Reveals a Common Progenitor for Endothelial, Myeloid, and Lymphoid Precursors in Umbilical Cord Blood. American Heart Association; September, 2010: pgs 1460-1468. 6. Reza, H. M ., et al. (2010). Characterization of a Novel Umbilical Cord Lining Cell with CD227 Positivity and Unique Pattern of P63 Expression and Function. Stem Cell; December, 2010: pgs. 1-15. 7. Fan, C. G., et al. (2010). Therapeutic Potentials of Mesenchymal Stem Cells Derived from Human Umbilical Cord. Stem Cell; July, 2010: pgs 195-204 8. Weiss, M. L., et al. (2005). Human Umbilical Cord Matrix Stem Cells: Preliminary Characterization and Effect of Transplantation in a Rodent Model of Parkinson’s Disease. Stem Cells; October, 2005: pgs. 781-792. 9. Yoo, K. H., et al. (2010). Current status of Pediatric Umbilical Cord Blood Transplantation in Korea: A Multicenter Retrospective Analysis of 236 Cases. American Journal of Hematology; September, 2010: pgs 12-17. 10. Choi, S., et al. (2011). HDAC Inhibition and Graft-versus-Host Disease. Mol Med; January, 2011: pgs 1-22