Umbilical Cord Blood

The following sections are included:

• Contents

• Contributors

• Preface

Hematopoietic stem cells (HSC) are among the best studied somatic stem cells. Studied for over 100 years and used in the clinical setting since over 50 years, HSCs are the only stem cells today that are transplanted in a routinely manner. In recent years it became apparent that in addition to bone marrow (BM), HSCs can also be obtained from umbilical cord blood (UCB). UCB-HSCs are thought to be more primitive than HSCs in the bone marrow, and the concentration of HSCs in UCB is higher than in BM. HSCs maintain the life long generation of blood cells of an organism and are essential for survival. And although HSCs are studied for so many years and are very well characterized, compared to other somatic stem cells, it is still difficult to grow HSCs in culture. Not surprisingly, UCB has become a major source of human HSCs for research, as UCB is more readily available than BM to most researchers. UCB has thus enabled the generation of a large body of research into the properties of these important human stem cells. The following chapter gives an introduction to the properties of HSCs and summarizes current knowledge about HSCs, particularly of UCB-HSCs, including phenotype, approaches to characterize their potential, and the specifics of UCB-HSCs.

Umbilical cord blood is a compelling alternative to bone marrow necessitated by the lack of HLA-matched unrelated donors and made possible by the recent proliferation of banking facilities. In this chapter, we discuss the clinical features of umbilical cord blood as observed over 20 years of transplantation for the treatment of hematologic malignancies and summarize several important clinical studies in pediatric and adult recipients. These studies have revealed a particularly low risk of high grade (III–IV) acute graft-versus-host disorder in cord blood recipients, slower myeloid recovery and the importance of strategies for augmenting cell dose, particularly in adult recipients. Current treatment protocols therefore indicate double unit cord blood transplants, which have demonstrated safety following myeloablative and non-myeloablative conditioning regimens. Studies probing the various cellular and molecular bases for these advantages comprise a field of intense inquiry and continue to contribute to the attractiveness of cord blood as a source of allogeneic hematopoietic stem cells.

The different mammalian blood cell types originate from hematopoietic stem cells (HSCs). HSCs are required in order to sustain the daily production of hundreds of millions of different blood cells fulfilling different functions. Since HSCs have been used for clinical applications for more than three decades, they represent the best studied somatic stem cell entity so far. However, the mechanisms controlling HSC maintenance or differentiation remain largely unknown. Focussing mainly on the process of asymmetric cell division, this chapter will review some of the current knowledge about the mechanisms controlling the decision “self-renewal versus differentiation” of primitive human hematopoietic cells.

Umbilical cord blood (UCB) provides an alternate source for patients undergoing high dose chemotherapy for treatment of cancer or genetic diseases. In particular, UCB has become a standard therapeutic option for selected patients with hematologic malignancies. Several studies have reported on the use of UCB for transplantation in adult patients, however, low cell doses per cord blood unit have limited the use of CB in this setting due to subsequent delays in engraftment. Ex vivo expansion is one approach that is being explored as a means to provide larger cell numbers from individual UCB products. In addition, recent clinical trials have demonstrated the potential to combine two UCB products to increase the cell dose delivered, however the majority of UCB products with sufficient cell dose, have a one or more antigen mismatch to each other or the recipient. Better matched products are available and ex vivo expansion may increase the cell doses for these products enabling better matched grafts.

This chapter will present an overview of past clinical trials and the approaches currently being explored.

Mesenchymal stem cells (MSCs), sometimes referred to as marrow stromal cells or multipotential stromal cells, represent a class of adult progenitor cells capable of differentiation to several mesenchymal lineages. A number of different names for MSCs describe similar but perhaps not identical multipotential cells of mesenchymal origin isolated in different labs — mesenchymal stem cells, mesenchymal progenitor cells, multipotential mesenchymal stromal cells, and others — but the abbreviation MSCs continues to be used by each of these laboratories. They can be isolated from many tissues although bone marrow has been used most often. MSCs isolated from different species from rat through man have very similar properties. MSCs may prove useful for repair and regeneration of a variety of mesenchymal tissues such as bone, cartilage, muscle, marrow stroma, and MSCs produce beneficial growth factors and cytokines that may help repair additional tissues. There is also evidence for their differentiation to non-mesenchymal lineages. MSCs interact with cells of the immune system in ways that allows them to be transplanted across immunological barriers and this is the subject of subsequent chapters. This chapter will provide a brief background on the origins of MSC research.

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. These standard techniques face significant disadvantages. As a result, research has focused on the development of alternative therapeutic concepts aiming to design and engineer unparalleled structural and functional bone grafts. Substantial academic and commercial interest has been sparked in bone engineering methods to stimulate, control and eventually replicate key events of bone regeneration ex vivo. Over the years, this interest has further increased and bone tissue engineering has now become a well-recognized research discipline in the area of regenerative medicine. The following chapter gives an overview of bone tissue engineering principles. It focuses on research related to the combination of scaffolds with multipotent precursor cells, such as bone marrow-derived mesenchymal stem cells or human umbilical cord perivascular cells, and the clinical applications of these tissue engineered bone constructs.

Stem cell and progenitor cells derived from the bone marrow and the cord blood have been shown to possess multilineage differentiation potential such as hematopoietic, mesenchymal as well as neural differentiation capabilities. This review summarizes recent advances in the field of neural differentiation in vitro and applications of cord blood derived cells in neurological preclinical disease models as well as in clinical applications in neurological diseases.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have generated great interest due to their ease of isolation, propagation and their ability to differentiate. Umbilical cord blood has been useful as a source of hematopoietic stem and progenitor cells, and investigators have sought to isolate umbilical cord blood (UCB-) or cord tissue-derived MSCs (UC-MSCs). These appear to be similar to BM-MSCs in their propagation, ability to differentiate and their immunological properties, suggesting they will be useful for tissue repair and transplantation. Subtle differences may exist that could be useful in tissue regeneration strategies. Initial efforts to isolate MSCs from bone marrow anticipated they would have immunological properties similar to hematopoietic stem cells, stimulate T-cell proliferation and be rejected by allogeneic T-cells. However, when tested in the mixed lymphocyte reaction (lymphocytes to MSC ratio of ˜10:1) MSCs failed to stimulate proliferation of T-cells. If MSCs were genetically engineered to express the missing co-stimulatory surface molecules, then they became immunogenic and stimulated the allo-T-cell response, but still not as actively as most cell types. Many researchers demonstrated that MSCs could actively suppress immune cell responses. MSCs express a number of molecules including hepatocyte growth factor (HGF), transforming growth factor β (TGF-β), prostaglandin E2 (PGE2), indoleamine 2,3- dioxygenase (IDO), nitric oxide, interleukin-10 (IL-10), IL-1 receptor antagonist (IL-1Ra), galectin-1 (Gal-1) and HLA-G that modulate the activity of the adaptive immune cells including inflammatory T_Helper1 (Th1), dendritic cells 1 (DC1), responding B-cells, as well as anti-inflammatory regulatory T-cells (T_Reg), T_Helper2 (Th2) and dendritic cells 2 (DC2). MSCs also have demonstrable interactions with the Natural Killer (NK) cells of the innate immune response. Animal studies support the notion that MSCs are immune privileged, although engraftment of transplanted MSCs remains low and leaves certain questions unanswered as to their universal transplantability or their best preparation for transplantation. Nevertheless, promising clinical trials have begun with BM-MSCs, and are in the planning stages with UC- or UCB-MSCs.

Mesenchymal stem cells (MSCs) are considered as emergent “universal” cells and various tissue repair programs using MSCs are today in development. The use of MSCs in such therapeutic strategies requires a large number of expanded cells and relies on their ability to maintain their own multilineage differentiation potential. MSCs expansion up to now almost completely relied on open preparations with multiple cell culture flasks. Moreover, these production processes are based on the use of fetal calf serum (FCS) and exogenous growth factors such as PDGF, FGF-2 or IGF-1 in an attempt to increase MSC proliferation rate. Today, a major challenge in producing cells for clinical use is the need to implement GMP compliant production processes to satisfy regulatory requirements. Some strategies are developed in this way using single-use materials like blood bags and transfer tubing, as well as closed culture devices validated in transfusion medicine. Furthermore, many studies demonstrated the advantage to replace FCS and exogenous recombinant growth factors by human platelet-derived growth factors.

It has been shown that platelet-derived growth factors were more efficient for ex-vivo expansion of MSCs than FCS: they promote MSC proliferation rates and decrease the time required to reach confluency. This completely closed process in serum-free conditions abrogates most safety concerns regarding microbial and prion contamination, which represents the major risk in autologous cell transplantation.

In the present chapter, we first describe the prerequisites for MSC isolation, culture and in vitro expansion: various sources of collection and different technical procedures of culture are discussed. Then the regulatory restrictions for a clinical-scale production of MSCs are detailed based on the French experience (traceability of ancillary products, closed medical devices, safe area, quality controls). Finally, an innovative technical procedure of clinical scale MSC production using clinical grade platelet lysate is described and production data are compared to the standard procedure using FCS and FGF-2.

Although unrestricted somatic stem cells (USSCs) from cord blood show a very homogeneous immunophenotype even after extensive amplification in vitro, our functional data suggest heterogeneity and the presence of a hierarchy of cells within such USSC populations and other non-adherent CD45⁻ populations. Certain USSC preparations reveal multipotency, associated with the lack of markers of senescence, long telomeres and high proliferative capacity. Others, in contrast, show a more restricted differentiation potential e.g., the cells can only form cell types of the mesenchymal lineage and are also limited in expansion capacity. We were able to define functional and transcriptome markers to dissect the hierarchy of USSCs and MSCs in cord blood and to highlight differences with bone marrow mesenchymal stroma cells. Based on these characterizations, USSC lines were generated and cultivated under GMP-conditions achieving 1.5 × 10⁹ cells in passage 3 to 4 corresponding to ten to 20 population doublings. Possible clinical and preclinical applications towards cardiac regeneration and support of hematopoiesis are the focus of this chapter.

The initial isolation of endothelial precursor cells (EPCs) over ten years ago has led to vast amounts of research and literature regarding these cells and their capabilities in the vascular system to contribute to new vessel formation. EPCs are endothelial progenitors that express combinations of CD34, VEGFR-2 and CD133. These EPCs differentiate into mature endothelial cells in vitro with endothelial functionality. In addition, EPCs have the capacity to be expanded in vitro enabling the generation of adequate quantities of this rare circulating cell. Various animal models of peripheral and myocardial ischemia have been utilized to show the effectiveness of EPCs in increasing perfusion, reduction of ischemic tissue and some incorporation of cells into new vasculature. It has been shown that EPCs diminish in number and function with age. However, future translational therapies with EPCs would likely involve an older patient population. Therefore umbilical cord blood may provide a source of younger EPCs. Studies show that UCB-derived EPCs have equivalent functionality to those derived from bone marrow or peripheral blood. With such potent capabilities, additional investigation into the potential of EPCs as cellular therapy for augmentation of vascularization and the use of UCB as a readily available source of EPCs are warranted.

The umbilical cord is a source of therapeutic cells. As described in other chapters in this book, umbilical cord blood is used clinically for reconstitution of the hematopoietic system following a number of diseases such as cancers, and certain congenital or metabolic diseases. We suggest that mesenchymal stromal cells derived from the umbilical cord may be useful therapeutic tools for a variety of diseases and for tissue engineering. While mesenchymal stromal cells are found infrequently in umbilical cord blood, they are found in abundance in the loose connective tissue surrounding the umbilical vessels, i.e., Wharton's jelly. In this chapter the characterization of Wharton's jelly-derived mesenchymal stromal cells, their status of banking for clinical evaluation, and preclinical work performed with these cells are reviewed and discussed.

The storage of publicly banked cord blood (UCB) units could eventually allow every patient who needs a hematopoietic stem cell transplant (HSCT) access to life-saving blood stem cells because of enhanced ability of UCB transplantation (CBT) to tolerate HLA mismatches compared to bone marrow transplantation (BMT), where stricter HLA matching requirements exist. Enhancements in CBT protocols, double CBT, ex vivo UCB expansion and homing technologies, together with a special effort to bank the higher cell dose units could overcome the problem of low cell dose for larger patients and adults. A worldwide network of UCB banks currently exists to help ensure that patients of different ethnicities in need of HSCT have maximal access to matching UCB cells, regardless of which part of the world they are in. Strict regulations in UCB collection, testing, processing, storage and distribution exist to protect the interests of both the transplant patients and UCB donors. More than 15,000 CBT have been performed around the world, using UCB units from public UCB banks. The storage of UCB units for personal use by private UCB banks, while rarely used and not encouraged by organizations like the American Academy of Pediatrics (AAP), is a personal decision made by families based on the remote likelihood of usage and the possibility that science could evolve in future to make such treatments more commonplace. Meanwhile public UCB banks, which provide the vast majority of UCB units for transplantation, have been serving as a common resource for the population for an increasing number of HSCT. Community and governmental support to increase the inventory of these public banks is critical to ensure the growth of this vital resource.

Banking cord blood is increasingly becoming more popular in many countries in the world. Cord blood banks are set up as public banks or commercial entities that provide fee-based services. This chapter explores the commercial aspects of cord blood banking including the necessity of private banks, their business models, hurdles faced by private banks and the factors that make private cord blood banking a successful enterprise. The differences between public and private banks are also discussed. The business models of these banks will also be impacted by the technological advances in the field of cord blood stem cells. As the use of cord blood cells for treating non-hematological disorders develops, the scale and requirements of banking cord blood will also change and private banks need to adapt themselves to this changing environment. In summary, this chapter discusses unique issues faced in the commercialization of cord blood banking and how private cord blood banks are tackling these issues.

The following sections are included:

• Closing Remarks

• Index