Umbilical Cord Blood Research: Current and Future Perspectives

Abstract

Umbilical cord blood (UCB) banking has become a new obstetrical trend. It offers expectant parents a biological insurance policy that can be used in the event of a child or family member’s life-threatening illness and puts patients in a position of control over their own treatment options. However, its graduation to conventional therapy in the clinical realm relies on breakthrough research that will prove its efficacy for a range of ailments. Expanding the multipotent cells found within the mononuclear fraction of UCB so that adequate dosing can be achieved, effectively expanding desired cells ex vivo, establishing its safety and limitations in HLA-mismatched recipients, defining its mechanisms of action, and proving its utility in a wide variety of both rare and common illnesses and diseases are a few of the challenges left to tackle. Nevertheless, the field is moving fast and new UCB-based therapies are on the horizon.

WHY UMBILICAL CORD BLOOD?

New sources of stem/progenitor cells that can replace lost or diseased cells of the body are being sought due to tight government restrictions, availability, and ethical considerations regarding the use of embryonic and fetal tissues. Of these, bone marrow (BM) is the current gold standard source of hematopoietic progenitor cells (HPC) used to reconstitute blood lineages after myeloablative therapy in a number of malignant and nonmalignant blood diseases (21). Bone marrow transplantation was first performed in the 1960s and is currently the treatment of choice for more than 15,000 patients worldwide each year (1). Allogenic and autogenic marrow can reconstitute erythrocytes, platelets, granulocytes, T- and B-lymphocytes, macrophages, osteocytes, Langerhans’ cells, Kupffer’s cells, and microglia (1). However, only 30% of eligible patients have a matched donor enabling them to receive its life-saving capability (2). The most notable BM transplantation disadvantages include: 1) The average length of time from commencement of the donor search to procurement of BM cells and treatment is 135 days (38), 2) the cost of locating a donor and harvesting the cells is considerable, ranging from $25,000 to $50,000 (38), 3) there is a low availability of human leukocyte antigen (HLA)-matched donors with BM, which is crucial for histocompatibility and avoidance of graft-versus-host disease (GvHD) in this type of allograft, 4) the National Donor Marrow Program has a strong European bias, making it difficult to find sufficient matches for people of other descents (17), 5) BM recipients have a high incidence of viral infection (90%) (5), and 6) patients with malignancies are often unable to use BM autografts because of the risk of reinfection with tumorgenic cells.

Umbilical cord blood (UCB), a once discarded material, has shown both in the lab and clinically to circumvent a number of these BM transplantation complexities. UCB cell transplantation made its clinical debut in 1988 when it was used to successfully treat a 5-year-old child afflicted with Fanconi anemia (27). Subsequently, more than 6,000 UCB transplants have been performed worldwide, many of them with unrelated donors (4,41,69,76). Like BM, UCB use has thus far been limited to hematopoietic malignancies, marrow failure, and immunodeficiency disorders (Table 1), but current research suggests it may be a much more powerful clinical weapon. UCB’s relative cellular immaturity compared to adult sources suggests a potentially unrivaled degree of plasticity. Its use as an alternative to BM transplantation continues to grow as research better defines its composition, mechanisms of action, and broad therapeutic capacity. Table 2 illustrates the currently known advantages to the use of UCB over adult stem/progenitor cell sources such as BM and adult peripheral blood (APB).

Table 1.

Some Clinical Uses of Umbilical Cord Blood to Date

Infantile Krabbe’s disease (22) Epstein-Barr virus (32) Lysosomal and peroxisomal storage diseases (46) Diamond-Blackfan anemia (75) Various acute and chronic leukemias (55) Neuroblastoma (55) Non-Hodgkin’s lymphoma (55) Hodgkin’s lymphoma (45) Sickle-cell anemia (55) Cooley’s anemia (33) Hurler syndrome (23) Leukocyte adhesion deficiency (63) Evans syndrome (52) Osteopetrosis (61) Spinal cord injury (37)

Table 2.

Umbilical Cord Blood Advantages

Easy procurement. No risk or discomfort to donors. Low incidence of viral contamination (38.2%) compared to BM (5) Able to be stored at cryogenic temperatures indefinitely without significantly affecting cell viability. Immediately available and easily shipped. There is a high immune tolerance of UCB cells because they are unable to generate cytotoxic T-Lymphocytes, which respond to allogenic antigens. UCB cells are also unable synthesize the proinflammatory cytokines interferon-γ(IFNγ) and tumor necrosis factor-α(TNFα) (58). UCB has an abundant source (~1% of the mononuclear fraction) of both hematopoietic and nonhematopoietic stem/progenitor cells, which is comparable BM (64,65). There is a large number of colony forming unit-granulocyte macrophage (CFU-GM) compared to adult sources (10). Procurement and use are not associated with the same ethical considerations as embryonic and fetal tissues.

WHAT IS UCB COMPOSED OF?

The cellular component of UCB is primarily comprised of lymphocytes and monocytes (51). It has a comparable B-lymphocyte population and a lower absolute number of T-lymphocytes (CD3⁺) but a higher CD4⁺/CD8⁺ ratio compared to APB (29,51). UCB also has higher numbers of NK cells while lower numbers of CD56⁺ cytotoxic T-lymphocytes (18). UCB’s relative immaturity compared to adult cell sources is further classified as showing a higher proportion of immature T-lymphocytes (CB45RA⁺) and decreased numbers of mature memory T-lymphocytes (CD45RO⁺) (18,29). UCB cells also produce fewer absolute levels of cytokines than adult cell sources (27). Furthermore, of the mRNA that is expressed in UCB, the anti-inflammatory cytokines interferon-γ (INF-γ), interleukin (IL)-4 and IL-10 are more abundant than for the proinflammatory cytokine IL-2 (27). This lack of mature immune function is attributed to UCB’s low incidence of GvHD and viral transmission. Such cellular constitution could allow for less stringent donor–recipient matching requirements, hence leading to shorter waiting period for treatment. Rocha (57) found that GvHD incidence was significantly lower in children receiving UCB transplants compared to BM recipients when the source was from an HLA-identical sibling. Rocha (56) also demonstrated a lower GvHD incidence in unrelated HLA-mismatched UCB recipients compared to HLA-identical BM recipients.

The enthusiasm over UCB began when it was found to contain a large population of hematopoietic stem/progenitor cells compared to adult sources. These easily procured, low immunogenic sources of multipotential cells are thought to have the capability to become any type of cell in the body under specific conditions. Not only does the MNF contain roughly 1% CD34⁺ cells, a marker designated for its role in early hematopoiesis, but these cells appeared to be more immature than those found in BM. In general, the level of maturity of a cell is identified by the cell’s presence of or lack of a combination of cell surface antigens. For instance, the CD34⁺ population in UCB can be defined as more primitive than those found in BM because a higher proportion (4×) of them are negative for CD38, a marker for pre-lymphoid cells (12,17). Another subset of CD34⁺ cells found in relatively high numbers in UCB are the more primitive CD133⁺ cells. CD133⁺ cells have been identified in fetal brain and in this area are considered to be neural stem cells (NSC) (67,70). However, it is not yet known whether the CD133⁺ cells found in UCB are phenotypically and functionally identical to the NSC found in fetal brain.

A nonhematopoietic stem cell, the mesenchymal stem cell (MSC), has also been found in UCB; however, in much lower numbers than in BM (28,82). The MSC can give rise to such diverse phenotypes as osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells (astrocytes and neurons) (35,39). Identification of this cell population is challenging because it currently lacks a definitive phenotype as well as agreement on exactly which surface antigens designate this cell. Yang et al. (82) characterized the MSC as having positive markers for CD13, CD29, CD44, and CD90 and negative markers for CD14, CD31, CD34, CD45, CD51/61, CD64, CD106, and HLA-DR, while Robinson et al. (54) defined the MSC as positive for CD73, CD90, CD105, and CD166 and negative for CD31, CD34, CD45, CD80, and HLA-DR. Universal agreement on a phenotype of these cells needs to first be reached before their true abundance in UCB can be known. Regardless, it is of a general consensus that there are far fewer MSC in UCB compared to BM (77).

EXPANSION NECESSITY

Because of the nature of the source there is but a single opportunity to collect UCB cells and, currently, the number of mononuclear cells (MNC) extracted from that isolated donation is finite. An average procurement contains only 1.5 × 10⁹ cells, which constitutes only 10% of a typical adult dose (adults represent 85% of all BM transplant recipients) (6). Furthermore, the standard method by which the MNC are obtained and stored results in a significant loss of the HPC population; in spite of this, for UCB banking to be fiscally sensible volume reduction is necessary (53). Among a number of different methods, extraction of MNC from whole UCB can be done through density gradient centrifugation using Ficoll (9). The MNC can then be stored at cryogenic temperatures in minimal space, unlike whole blood. Expansion is central to providing adequate dosing for both children and adults, as well as for recurrent therapy in the event of graft failure. Certain criteria need to be met when considering expansion of UCB cells. First, determining which cells should be expanded. As stated earlier, UCB contains a large population of CD34⁺/ CD38⁻ and CD34⁺/CD133⁺ cells, but also important is its component of colony-forming unit (CFU) required for reconstitution of BM and expression of CXCR4 (stromal cell-derived factor-1) involved in homing of cells to the BM where repopulation occurs (19). Secondly, expanded cells also need to maintain telomerase length in order to preserve their pluripotent capacity (83). Telomerase activity is high in HPC and tends to be most active during proliferation (16). Table 3 lists a number of laboratories that have had varied success with ex vivo expansion of UCB MNC using different techniques and medias.

Table 3.

Current Expansion Success of Cord Blood Mononuclear Cells

Cell Type(s)	Expansion Fold	Notes
CD34+ GM-CFU	12 and 20, respectively	Culture using a closed cell expansion chamber (3).
CD34+, CFU	27 and 22, respectively	7 days in serum-free culture (83).
CD34+		IL-11 and G-CSF stimulated, created 80-fold greater yield than BM (31,68,71).
CD34−/CD45−	1015	No loss of pluripotency (39).
Total MNC, CD34+, CFU-GM	435.5, 32.7, and 21.7, respectively	200 h culture in a rotating wall vessel bioreactor was used with serum containing media supplemented with low-dose recombinant human cytokines (43).
CD34+, CD45+, CFU	3, 3, and 2.6, respectively	Serum-free, cytokine-free 3D cytomatrix (20).
CD34+, CD34+/CD38−, CFU-C	250	Expanded on a monolayer of human BM MSC with cytokines and serum (81).
MSC	1000	In culture with serum for 3 weeks (82).

The primary purpose of expansion is to reduce the time to engraftment, thereby increasing treatment success. Therefore, an important consideration for expansion of UCB must be the inclusion of neutrophils and platelets. Safety has been established using expanded UCB cells in patients, but the time to neutrophil and platelet engraftment has not successfully been reduced (34,62). The low antigenic response of UCB cells is its driving strong point, leading to lower incidence of GvHD, but it also poses a significant problem. The time to engraftment is delayed compared to BM due to UCB’s more primitive, nonnucleated stem/progenitor cell component, which requires more time to reconstitute platelets and infection-fighting granulocytes.

Global expansion of the MNC from a typical UCB unit is not the greatest technical feat. Though the correct cell/kg body weight dosage is important, it is the number of UCB cell subpopulations that dictates effective, timely engraftment. Current methods of expanding UCB cells do not preserve the quality of the HPC through to the end product (34,54). Recovery of the CD34⁺ cells from frozen units of UCB is also poor (47). Current expansion methods cannot make up for the cells lost in the storage process (31,43,68,71,83), let alone augment them to a suitable dosing regimen.

A need for optimizing culture media that maintain the primitive nature of the desired subpopulations is needed. Some have demonstrated that coculture with MSC as support cells may preserve immature, repopulating cells (8,50,54). MSC coculture is advantageous in that no isolation of the UCB HPC is required, curtailing superfluous loss of cells (54). However, the MSC used to aid expansion of UCB HPC are typically of a xenogenic origin, generating the possible introduction of infectious diseases to the patient (81). Use of an allogenic stromal layer would be difficult due to the extreme rarity of MSC in UCB (7,28).

Expansion challenges keep widespread UCB transplants in clinical infancy. Simply multiplying the cells globally has proven not to decrease time to engraftment (34,62). Instead, relative proportions of cell subpopulations that home to marrow, form colonies, and reconstitute blood lineages are required. This key clinical obstacle remains elusive to researchers. Continued investigation will unveil the true therapeutic potential of UCB for reconstitution of lost or diseased cells.

INITIAL ENTHUSIASM OVER UCB

Clinical applications of UCB to date have focused on hematologic reconstitution. But research suggests multipotent cells in the heterogeneous MNC population may differentiate into osteoblasts, chondroblasts, adipocytes, and even neurons and astrocytes (39). Because of the high proportion of stem/progenitor cells in UCB the thought was that it may substitute dependence on use of the highly controversial embryonic and fetal stem cells for replacement therapy in a variety of disorders. Several reports have been published claiming the multipotent nature of UCB cells when directed under the right conditions.

Buzanska et al. (11) used the CD34⁻/CD45⁻ nonhematopoietic MNC fraction of UCB to obtain neural stem-like cells. From these, a clonogenic line of human UCB-neural stem cell (NSC), exhibiting immunophenotypic markers for nestin and glial fibrillary acidic protein (GFAP), was derived. In the presence of neuromorphogen/retinoic acid (RA) 40% of the human UCB-NSC expressed βIII tubulin and MAP-2, 30% expressed the astrocytic markers GFAP and S100β, and 11% expressed the oligodendrocytic phenotype, galactosylceramide (GalC). RA brain-derived neurotrophic factor (BDNF) has also been shown to induce neural differentiation in human UCB-NSC culture. During 7 days of co-culture with neuromorphogens, rat astrocytes, or hippocampal slices, 80% of cells expressed βIII-tubulin and 64% coexpressed microtubule associated protein (MAP)-2, a marker for advanced neuronal differentiation (36). Also, Xiao and colleagues have produced a line of cells isolated from UCB that they term nonhematopoietic umbilical cord blood stem cells (80). A reduction in infarct volume was observed after intravenously transplanting these cells into rats with ischemic brain injury. Histological analysis revealed that some of the transplanted cells were double labeled for human nuclei and NeuN, though it was unlikely that they contributed to the recovery.

Our group has induced expression of neural proteins in UCB MNC. Using a neural proliferation medium consisting of serum-free Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and N2 (used for neural proliferation) and a differentiation medium of DMEM/F12 and N2 supplemented with RA and nerve growth factor (NGF), Sanchez-Ramos et al. (59) persuaded expression of Musashi-1, TuJ1, βIII-tubulin, and GFAP. Zigova et al. (84) took the work a step further, showing that TuJ1 and GFAP immunopositive cells from UCB MNC after treatment with RA + NGF survived in the subventricular zone (SVZ) of the rat neonatal forebrain. More recently, Chen et al. (15) conducted characterization analysis of the cell subpopulations within cultured UCB, finding neurotrophin receptors in both floating and adherent fractions. Exciting though this may seem, what is not yet known is whether these in vitro-produced, phenotypically similar cells function as their in vivo-derived counterparts. Establishing appropriate function will be crucial before a line of UCB cells can advance toward replacement strategies in the clinic.

CURRENT RESEARCH

Because of its large population of HPC, UCB was thought to be the ideal source of cells to be used for replacement of dead and/or diseased cells in a number of injuries and diseases. In vivo research has found that human UCB can ameliorate behavioral and physiological consequences in a number of animal disease models, the most exciting of which included diseases and injury of the brain. Our lab and others have demonstrated UCB’s growth as a multidimensional treatment (Table 4).

Table 4.

Preclinical Successes Using Umbilical Cord Blood to Treat a Variety of Animal Models of Disease and Injury

Amyotrophic lateral sclerosis (13,26) Sanfilippo syndrome type B (24,25) Spinal cord injury (40,60) Traumatic brain injury (44,48) Stroke (14,49,72,73,78–80) Myocardial infarction (30,42)

Linking each disease model with recovery facilitated by the use of UCB is the lack of defined mechanism(s). Clearly, UCB works uniquely depending on its environment and the nature of the injury—a presumed molding to fit the need. The multifaceted nature of UCB’s therapeutic effects is no doubt a function of its heterogeneous make up, and also is what eludes researchers attempting to classify its utility. Of course, this is the goal and the necessity before UCB can achieve widespread use in the clinic so that the question of safety can be adequately addressed.

New research in our lab has found that UCB may not only act as a cell replacement source, but also as a neurotrophic, neuroprotective, and anti-inflammatory agent. The most notable example of the multifaceted therapeutic effects of UCB is in the middle cerebral artery occlusion model of embolic stroke. In this rat model we have achieved profound behavioral improvement and infarct reduction. Stroke involves a complicated cascade of inflammatory events that eventually lead to a pronounced area of cell death adjacent to the blocked vasculature. This cascade is time dependent and UCB, when given intravenously 48 h following stroke onset, can reverse impending cell death. The temporal administration of UCB most likely inhibits the apoptotic cascade, and modulates the immune/inflammatory response to injury both peripherally and locally (49,73,74). UCB treatment also decreases the number of CD45⁺/CD11b⁺ and CD45⁺/B220⁺ cells and the proinflammatory cytokines, TNF-α and IL-1β, in the brain following stroke (73), as well as expression of both activated microglia and astrocytes (49). The potential anti-inflammatory effects of UCB therapy may protect against neuronal death while evidence suggests that the CD34⁺ component of the transplant may facilitate revascularization (66). Essentially, UCB provides an arsenal of therapeutic effects in a single transplant that no pharmacological agent could mimic.

Although we use stroke here to illustrate the multimodal properties of UCB therapy, it is by no means limited to stroke injury. A multitude of diseases exhibit apoptosis, an inflammatory component and need for neuroprotective therapy. The therapeutic potential of UCB is far reaching from common ailments to exotic diagnoses. If discovering its mechanisms of action to ensure safety in its use and perfecting the expansion process is all that remains then its widespread penetration into hospitals and clinics is inevitable.