Lifeline in an EthicalQuagmire: Umbilical

Cord Blood as anAlternative to

EmbryonicStem Cells

Ian Rogers, Ph.D.

Robert F. Casper, M.D.Division of Reproductive Sciences,

Department of Obstetrics and Gynecology, University of Toronto,and Samuel Lunenfeld Research Institute,

Mount Sinai Hospital

Embryonic stem (ES) cells are capable of unlimited self-renewal and have the ability togive rise to all tissue types in the body. Recently, tissue-specific stem cells such as bonemarrow cells have also been found to be capable of multilineage differentiation into cells

of various nonblood tissues. Umbilical cord blood hematopoietic stem cells have beenshown to be as effective as bone marrow stem cells for rebuilding the hematopoietic

system and differentiating into nonblood cell types. This observation raises the excitingpossibility of replacing human ES cells for tissue and cell therapeutics with umbilical cord

blood hematopoietic stem cells that are normally discarded with the placenta afterdelivery.

TECHNOLOGY

• What if the umbilicalcord blood stemcells we usuallydiscard with theplacenta couldreplace controversialembryonic stem cellsin therapy?

Sexuality, Reproduction & Menopause, Vol. 2, No. 2, June 2004© 2004 American Society for Reproductive Medicine

Published by Elsevier Inc.

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Stem cells are a complex cell typethat is not easily defined. Theaccepted definition of a stem cellis a cell that is capable of devel-oping into multiple cell types

and having unlimited self-renewal; how-ever, only in vitro grown murine embry-onic stem (ES) cell lines fit this definition(1). This review provides evidence thatumbilical cord blood hematopoietic stemcells are likely to replace ES cells in thefuture for tissue therapeutics, therebyavoiding the ethical and practical prob-lems surrounding human ES cells.

Embryonic stem cells were first developedfrom the inner cell mass (ICM) of the mu-rine blastocyst (2). These cells representthe ultimate stem cell because of their abil-ity to self-renew, be multipotent, and con-tribute to all germ layers (3), something thefounder cells of the inner cell mass cannotdo in situ.

Human embryonic stem cells act similarly to murine embryonic stem cells, with some minordifferences. Both cell types require a feeder layer of embryonic fibroblasts to remain undiffer-entiated, but human ES cells may require the addition of basic fibroblast growth factor (bFGF)or leukemia inhibitory factor (LIF). Embryonic stem cells have been tested for gene expressionby polymerase chain reaction (PCR) and they have been grown in specific cultures to determinetheir ability to form a wide range of tissues. Both types of ES cells will form embryoid bodiesthat resemble a blastocyst and acquire characteristics of the endoderm, mesoderm, andectoderm. Human ES cells can form beating myocytes, neuron-like cells, and hematopoieticcells (4).

The therapeutic use of human embryonic stem cells to replace damaged tissues or organs is anexciting, although controversial, goal. A possible alternative source of therapeutic cells in-cludes tissue-specific stem cells, which to some extent have some properties similar to those ofembryonic stem cells but are less able to turn into other cell lines. Because they come frommultiple sources, each with its own unique potential, however, their combined ability mayprovide a full spectrum of tissue therapies and thereby may eliminate the need for human EScells in the future.

One well-studied tissue-specific stem cell, the hematopoietic stem cell (HSC), is able to makenew blood cells for the life of the organism, but has only a limited ability to differentiate intoother types of cells (5). Many other adult tissues possess a subset of cells with stem cellproperties. Examples are neural stem cells (6), which can self-renew only neural cells; musclestem cells (satellite cells) (7); and hepatic stem cells (oval cells) (8).

Hematopoietic stem cells were thought to be preprogrammed during embryo development, butenvironment and internal genetic programming may play roles as well. Multipotent cells of theearly primitive streak from mouse embryos will reproducibly contribute to specific tissues, butif more differentiated cells of the late primitive streak are transplanted, they can differentiateinto various host tissues depending on the specific transplant site (9). It appears, therefore, thatsome predetermination exists, but a degree of ability to respond to signals from surroundingcells is retained during passage through the primitive streak (10).

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Tissue-specific Stem Cell Differentiation

At first glance, it seems surprising that tissue-specific stem cells may also have the ability to bemultipotent. Lineage restriction of cells may be an important mechanism that has developedover time as a way to ensure reproducible development of the embryo and that developmentalrestriction occurs due to cell response to external signals from neighboring cells rather than byinherent limitation of the potential of the tissue stem cells.

Can tissue-specific stem cells be altered to produce a self-renewing multipotent cell similar toan ES cell? In vivo studies using clonal cell populations or single cells strongly suggest that theanswer is yes. However, more studies are needed to verify whether or not tissue-specific stemcells can replace damaged or diseased tissue with functional cells.

To use adult stem cells safely for tissue therapy, we must first determine the mechanism(s) ofstem cell differentiation. Do stem cells respond to signals involved in tissue healing? WhenHSCs are transplanted into severe combined immunosuppressed (NOD/SCID) mice, the recip-ient mice are irradiated, resulting in tissue damage to the bone marrow, liver, and other tissues.The cell signaling in the damaged tissue (e.g., liver) that triggers cellular repair mechanismsmay also trigger the differentiation of HSCs to the liver phenotype. There has recently beensuccess in using hematopoietic stem cells to treat a mouse model of Parkinson’s disease.Parkinson’s disease occurs due to the loss of dopamine-expressing neurons in the substantianigra due to cell death. It is possible that repair signals from the surrounding neurons maytrigger differentiation of HSCs, which find their way to the substantia nigra after transplanta-tion, into dopamine-producing neurons (11).

An alternative mechanism could be cell fusion rather than transdifferentiation. In other words,cells don’t actually transform, but rather fuse with another cell and take on its characteristics.Recently, neural stem cells cultured together with a muscle cell line (C2C12 cells) resulted inthe conversion of the neural stem cells to muscle, but this conversion occurred only if theneural stem cells were in contact with the C2C12 cells. Clusters of neural cells separated fromC2C12 cells within the culture failed to generate muscle characteristics. This conversion wasirreversible; once the neural stem cells differentiate toward mature muscle cells they becomeunable to differentiate back into neural cells (12). Regardless of whether the stem cell fuses ortransdifferentiates, the ultimate result is still repair of the damaged tissue.

Clinical Evidence for the Multipotential of HSCs

In an interesting clinical study, donor bone marrow cells became functional hepatocytes in therecipient’s liver tissue after transplantation. This was a retrospective study in which the bonemarrow transplant recipients and the donors were of different sex. Using a combination of sexchromosome-specific fluorescence in situ hybridization (FISH) combined with immunohisto-chemistry for liver-specific markers, it was possible to clearly show that bone marrow cellscontributed to functional liver cells within the recipients (13). The recipients received highdoses of radiation to deplete their bone marrow of cells; it is likely that the radiation causedsome hepatic injury at the time of bone marrow grafting. This may have set the stage forappropriate signaling from the damaged tissue to result in transdifferentiation of the bonemarrow stem cells to hepatocytes. Alternatively, the mechanism of hepatocyte formation mayhave been cell fusion. Careful studies using donor- and recipient-specific cell markers may helpto determine which of these mechanisms is active.

Therapeutic Cloning

The birth of the cloned sheep Dolly in 1997 suggests that a terminally differentiated adultsomatic cell nucleus, reprogrammed using somatic nuclear transfer (SNT), can cause a tissue-specific stem cell to transdifferentiate (14). Recently, Hochedlinger and Jaenisch (15) usedB-cell nuclei in an SNT experiment to generate functional ES cells. These two studies provethat a terminally differentiated adult cell can be reprogrammed to produce multiple cell types.

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Therapeutic cloning has some attractiveness since it avoids the problem of tissue rejection dueto HLA mismatches. However, clonally derived embryos may not be normal in differentaspects, including epigenetic stability and telomerase function (16, 17), and will not reach theblastocyst stage to allow embryonic stem cells to be produced from an inner cell mass. Anadditional concern is that human oocytes are currently necessary for SNT. Both problems willlikely make therapeutic cloning impractical if not unattainable.

Bone Marrow-derived Adult Stem Cells

Bone marrow-derived stromal and hematopoietic cells are able to differentiate into multiplecell types, thereby demonstrating ES cell-like properties. Hematopoietic stem cells purifiedfrom bone marrow and transplanted into recipient mice can differentiate into hepatocytes (18)and rescue a liver defect, demonstrating functionality. In another study, HSCs were purifiedand clonal populations examined to determine if they could self-renew or differentiate.Long-term repopulation of irradiated hosts showed that these cells not only migrate to the bonemarrow, but also can differentiate into epithelial cells of the liver, lung, gastrointestinal tract,and skin, albeit in small numbers (19). Mesenchymal progenitor cells from the stromal cells ofthe bone marrow also have the ability to produce many diverse cell types from a clonalpopulation, both in vivo and in vitro (20).

Are Umbilical Cord Blood Hematopoietic Stem Cells Multipotent?

Umbilical cord blood was first used for a successful bone marrow transplant in a patient withFanconi’s anemia in 1988 (21). Hematopoietic stem cells are found in the fetal circulation, andin the 100 mL or so of blood in the placenta and umbilical cord, which are typically discardedafter delivery. Within hours following delivery, the HSCs migrate to the bone marrow wherethey provide the progenitors of all the blood-forming elements, including erythrocytes, leuko-cytes, and platelets.

The main focus of our laboratory is to determine whether cells from umbilical cord blood havesimilar properties to human ES cells. In preliminary experiments, human umbilical cord bloodCD34� cells were grown in vitro in serum, showed mesenchymal cell morphology, and werepositive by PCR for bone (TRAP), muscle (desmin), neural (nestin), and astrocyte (Gfap)markers. The cells also had positive staining for the vimentin antibody, confirming themesenchymal morphology. These results demonstrate the multiple cell potential of umbilicalcord blood cells and confirm previously reported findings.

Cord Blood Stem Cell Expansion

There are enough hematopoietic stem cells in an umbilical cord to replace the bone marrow ofa child; only 25% of stored samples in our bank contain sufficient numbers of cells totransplant an adult. Stem cell expansion in vitro, therefore, is an important goal, not only forincreasing the use of cord blood HSCs for replacing bone marrow transplants, but also for tissuetherapeutics. Having enough cells to make clinical therapeutics possible will be a major hurdleto overcome. Hematopoietic stem cells will divide, producing one daughter stem cell and aprogenitor cell, which will then produce mature blood cells. Reproducing this division processin vitro will only be sufficient to maintain levels of the starting population. However, it isdifficult to cause hematopoietic stem cells to proliferate because the process also causesdifferentiation, as growth factors have both mitogenic and differentiation properties. The keybarrier to in vitro cell expansion, therefore, is the loss of self-renewing stem cells that occursduring induced cell proliferation. For one stem cell to give rise to two new stem cells, thedifferentiation pathway must be blocked by triggering proliferation before the onset of the cells’internal differentiation program (22).

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Efficiency of Differentiation of Various Types of Stem Cells to Mature Cells

Although the percentage of cord blood HSCs demonstrating nonblood markers was low in ourexperiments, other types of stem cells have a surprisingly consistent low rate of differentiationto mature cells. Murine embryonic stem cells have the highest efficiency of production ofspecific mature cells among all stem cells. Dang et al. (23) were able to increase the productionof embryoid bodies to 42% with a continued differentiation to hematopoietic cells (as mea-sured by hematopoietic colony forming unit [CFU]) to about 5% of the input ES cells. Theconversion of murine ES cells to cells with neuronal markers was only 0.2% of the input EScells (24), while others (25) demonstrated that 1% of the cells originally isolated from day 9.5murine embryos produced neural spheres, with 20% of the primary spheres being capable ofproducing secondary spheres. Of these secondary spheres, 80% of the spheres are positive foroligodentricytes, neurons, and astrocytes; therefore resulting in a differentiation rate of lessthan 16% of the primary sphere population.

About 5% of skin cells isolated from a skin biopsy will proliferate in culture and demonstratethe ability to form neural cells (26). With mesenchymal cells from bone marrow, about 1/10,000of adherent mononuclear cells (CD45�) will produce a proliferating colony with multiplepotential. Of these isolates, when placed into differentiation cultures, 90% become endothelialor neural and 60% become albumin positive in cultures that favor hepatocyte development(20).

Our studies demonstrate that about 10% of our enriched population of umbilical cord bloodstem cells will proliferate in culture and demonstrate an expanded range of differentiation. Ofthese proliferating cells, 80% will be positive for neural, endothelial, muscle, or mesenchymalcell markers in vitro.

Stem cells from different sources, therefore, demonstrate similar rates of differentiation. Whatdistinguishes them from each other, as the prime candidate for cell therapeutics, is the abilityto proliferate sufficiently to overcome the low rates of differentiation. Currently, ES cells havethe most efficient proliferation in vitro but in the future, conditions for high levels ofproliferation of tissue-specific stem cells are likely to be developed. Our research has alreadyyielded a 15- to 20-fold increase in cord blood HSCs in culture; this increase is likely toimprove in the future.

The therapeutic use of human embryonic stemcells to replace damaged tissues or organs is anexciting, although controversial, goal. A possiblealternative source of therapeutic cells includestissue-specific stem cells, which to some extenthave similar properties to those of embryonicstem cells.

Ethical Issues

The use of human ES cells for tissue and cell therapeutics will ultimately be limited by ethicalconcerns since these cells are derived human embryos. The need for HLA matching of EScell-derived tissues for allogeneic transplantation will require the production and banking of

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several thousand ES cell lines to make tissue therapeutics practical. This requirement would,therefore, require tens of thousands of starting embryos since ES cell derivation from humanembryos is an inefficient process at present. We believe that a nonembryonic source of stemcells will help to overcome the ethical and practical issues associated with the use of humanembryonic stem cells. Of adult stem cell studies so far, umbilical cord blood hematopoieticstem cells are the most attractive because they are obtained from material that is generallydiscarded following delivery. The development of several thousand umbilical cord bloodsamples to address the HLA matching problem is a simple matter, limited only by the numberof deliveries in any particular region and the willingness of parents to donate cord blood.

Conclusion

Studies by our laboratory demonstrate that human umbilical cord blood hematopoietic stemcells appear to have the capacity to proliferate in vitro without differentiation, to acquiremarkers consistent with an ES cell phenotype, and to transdifferentiate into nonblood celltypes such as hepatocytes, neurons, and muscle. This observation raises the exciting possibilityof replacing human ES cells for tissue and cell therapeutics with umbilical cord bloodhematopoietic stem cells that are normally discarded with the placenta after delivery. Inaddition, the ease of creating umbilical cord blood banks containing tens of thousands ofsamples realistically ensures the ability to achieve appropriate HLA matching for recipients ofsuch therapy, in contrast to the practical and ethical concerns of creating sufficient ES cell linesfor proper HLA matching. We anticipate that future studies by ourselves and others willconfirm the clinical usefulness of umbilical cord blood HSCs in tissue therapeutics and genetherapy.

Robert F. Casper, M.D.,Division of Reproductive Sciences,

University of TorontoSamuel Lunenfeld Research Institute,

Mount Sinai Hospital,150 Bloor St W., Suite 210,

Toronto, Ontario, Canada, M5S 2X9e-mail: rfcasper@aol.com

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