umbilical cord blood stem cells

Best Practice & Research Clinical Obstetrics and GynaecologyVol. 18, No. 6, pp. 893–908, 2004

5

Umbilical cord blood stem cells

Ian Rogers PhD

Assistant Professor

Robert F. Casper* MD, FRCS

Professor

Division of Reproductive Sciences, Department of Obstetrics and Gynaecology, Samuel Lunenfeld Research Institute,

Mount Sinai Hospital and The University of Toronto, Toronto, Ont., Canada

The umbilical cord contains a rich source of haematopoietic stem cells that can be used toreconstitute the blood system and can easily be extracted and cryopreserved, thus allowing forthe establishment of HLA-typed stem cell banks. Recently, it has been demonstrated thatumbilical cord stem cells have the potential to give rise to non-haematopoietic cells, such as bone,neural and endothelial cells. It is not clear whether these multipotential cells are mesenchymal-like cells or blood cells. Currently, the number of these specialized cells capable of undergoing thedifferentiation process into non-haematopoietic cells is low and remains a block to the clinicaldevelopment of umbilical cord stem cells for non-haematopoietic cell therapy. Further researchwill allow us to overcome these hurdles. This expanded potential for umbilical cord stem cellsmight replace embryonic stem cells and other fetal cells for some cell and tissue therapies.

Key words: bone; endothelial; neural; stem cells; umbilical cord blood.

Over the past few years there has been a vast amount of information, from manydifferent laboratories, espousing the virtues of newly discovered stem and progenitorcells from a wide variety of tissues.1–4 The discovery of new sources of stem andprogenitor cells is in itself an amazing story, which has and will continue to turn themedical field on its ear. As discussed in Chapters 1 and 2, the modern paradigm for stemcells is the embryonic stem (ES) cell, first derived from the mouse5 and more recentlyfrom non-human primates6 and humans.7 The obvious dilemma facing scientists andclinicians interested in the development and use of human ES cells for widespread tissue

doi:10.1016/j.bpobgyn.2004.06.004available online at http://www.sciencedirect.com

1521-6934/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

* Corresponding author. Address: Division of Reproductive Sciences, Department of Obstetrics and

Gynaecology, Samuel Lunenfeld Research Institute, Room 876, Mount Sinai Hospital, 600 University Avenue,

Toronto, Ont., Canada M5G 1X5. Fax: C1-416-586-8588.

E-mail address: [email protected] (R.F. Casper).

http://www.sciencedirect.com

894 I. Rogers and R. F. Casper

therapy is the source of these cells: the human embryo. Although experiments usingmurine ES cells and recent experiments with human cell lines have demonstratedthe incredible potential of these cells, their intimate connection to the humanblastocyst will always be a source of concern. Similar ethical issues were raised withearlier research surrounding the use of therapeutic fetal tissue transplants, which bynecessity, were obtained following elective pregnancy terminations.

Fetal tissue transplantation

Fetal tissue transplantation was introduced as a research therapy for chronicdegenerative diseases such as Parkinson’s disease and insulin-dependent diabetes.Fetal cells were believed to have several advantages over adult cells because they canproliferate more rapidly, have lower levels of histocompatibility antigens and can surviveat lower oxygen tensions, which makes them more resistant to hypoxia duringtransplantation.8 However, in 1988 the United States Department of Health andHuman Services instituted a moratorium on the funding of research using fetal tissuefrom elective abortions because public concern surrounding fetal tissue transplantationwas perceived to be so great.9 Although this funding was restored in 1993, controversyconcerning fetal tissue transplantation remains. There are many opponents to thistherapy on the grounds that fetal tissue is obtained from elective abortions. Theseopponents believe that the perception of a positive use of fetal tissue could increase therate of abortion by making it easier for a woman to justify pregnancy termination. Inaddition, demonstration of a curative effect of fetal tissue transplantation could evenencourage some women to conceive in order to have an abortion to help a familymember with Parkinson’s disease or diabetes.10

More significant at the present time is the lack of data for efficacy of fetal tissuetransplantation for the uses described above. Recent data from a double-blind, placebo-controlled trial of fetal nigral transplantation for Parkinson’s disease showed nosignificant overall treatment effect.11 Fifty-six percent of transplanted patientsdeveloped dyskinesia within 12 hours of stopping their medication, although therewas a weak treatment effect in the most mildly affected patients.11 This lack ofeffectiveness might be the result of continuing degeneration of the host nigrostriatalsystem whereas any benefit from the engrafted tissue is a result of increased dopaminesecretion rather than improved dopaminergic innervation of the host striatum.12,13

In patients with labile type 1 diabetes, it appears that research with fetal islet celltransplantation has been replaced by adult islet cell transplantation as described byShapiro et al14 in Edmonton, Alberta, Canada. The so-called ‘Edmonton protocol’involves percutaneous transhepatic portal embolization of isolated islet cells from twoor three cadaveric adult donor pancreases together with a glucocorticoid-freeimmunosuppressive regimen. However, in follow-up, it appears that only about 20% ofpatients achieve insulin independence at 1 year.15

Embryonic stem cells

Similar ethical and emotional concerns exist over the use of ES cells in research, asthese cell lines are derived from the inner cell mass of human blastocysts. In the processof understanding and eventually developing stem cells from less controversial sources,e.g. blood or bone marrow, ES cells provide a useful model from which to deriveanswers to the complicated questions concerning self-renewal and regulated

Umbilical cord blood stem cells 895

differentiation. Limiting the number of stem cell lines available as done in the USA (seewww.nhlbi.nih.gov/funding/policies/escells.htm) or regulating the production and use oflines as proposed in Canada (see www.cihr-irsc.gc.ca/e/publications/1487.shtml) areattempts to address some of the issues surrounding ES cells and point to the problemsof balancing a potential life-saving therapy with moral and ethical concerns.

Embryonic stem cell research is still in its infancy and there is much to be discoveredabout these amazing cells. All this points to the fact that using ES cells in everydaymedicine is a long way off. Yet the power of these cells and the impact of thecontroversy regarding their genesis have led many scientists to look for alternativesources of stem cells. As a result, although ES cell therapy is in its infancy, its successmight result in it being superseded by alternative stem cell therapies.

Haematopoietic and other stem cells

Haematopoietic stem cells from the bone marrow have been used extensively for yearsto reconstitute the bone marrow of recipients suffering from a number ofhaematological disorders.16 The constant replacement of the haematopoieticcompartment throughout our lives had led to the search for and discovery of bloodstem cells.17 The discovery of stem cells specific to other tissues did not come untilrecently, probably because other tissues do not have the same ability of self-repair orregeneration as that of the haematopoietic system and, therefore, were not consideredlikely candidates as stem cell repositories. The discovery that the adult brain maintainedsome capacity to make new neurons1 was surprising to say the least, and led toquestions such as: (i) If stem cells exist in most tissues, do they contribute to repair ofthe host or other tissue? and (ii) If these cells do maintain stem cell properties of self-renewal and the capacity to differentiate into multiple tissue types, even at a minimallevel in situ, can they be coaxed into higher levels of activity through manipulation?

Haematopoietic stem cell transplantation

Haematopoietic stem cells from bone marrow are easy to isolate and can betransferred from the donor to the recipient with virtually no manipulation because thehaematopoietic system has a high rate of renewal normally. For any type of stem celltransplant, certain considerations must be taken into account by transplanters if theyare to obtain a successful graft. These factors include matching the HLA type of thedonor with that of the recipient, determining the critical numbers of stem cellsrequired, the use of accessory cells or factors, the extent and type of tissue-specificinjury, the time lapse expected between injury and transplant and the required rate ofrepair in order to successfully repair the injury. These considerations apply to all tissue-specific stem cell therapies, although some take on a different importance depending onthe type of stem cell being considered for transplant. Using the haematopoietic systemas an example, the number of cells infused correlates directly with the success of thetransplant as measured by platelet and neutrophil counts at 2–3 weeks post-infusion.The time element is an important consideration because, as shown experimentally, asmall number of cells is able to engraft and produce a full haematopoietic system butwill require a greater length of time than a larger cell number.18 Therefore, in theory,fewer cells can be used but too much time may elapse and the patient might die as aresult of secondary factors (infection or bleeding) related to the lack of a functionalhaematopoietic system. Furthermore, the length of the hospital stay and medication oraccessory therapies required to keep the patient alive during a prolonged recovery

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must be factored in. It has been shown that a single haematopoietic stem cell canregenerate the whole immune system in a mouse model but success was due to the factthat the endogenous system was still functional and the mouse survival was notdependent on the donor cell.19 Therefore, tissue-specific stem cell transplants that havelow cell numbers or a reduced capacity to proliferate might prove useless because timeis not on the patient’s side.

Another consideration for transplantation is the accessibility of tissue-specific stemcells. For example, neural stem cells might prove to have great capacity in the area ofneural regeneration but the obvious problems of retrieving these cells will placelimitations on future uses. Single stem cell lines, such as those of murine origin, aresuitable for research purposes but not clinical use. Tissue matching, as required for anyorgan transplant or bone marrow transplant, is expected to be required for all otherstem cell therapies. HLA matching is important and will require the production ofeither extensive databases with information on potential donors, as occurs with bonemarrow registries, or the establishment of large, HLA-diverse, cell or tissue banks.20–24

HLA-matched cell and tissue banks that would encompass all possible tissue-specificstem cells including haematopoietic, muscle, neural, bone and others would be aformidable endeavour, especially as neural cells would depend on sources similar to thatof organ donation.

Which stem cell sources are best suited for registries or cell banks is partiallydependent on their proliferation and self-renewal capacity. ES cell lines have unlimitedproliferation capabilities and survive cryopreservation, thus making them excellentcandidates for cell banking. Yet the creation of a complete ES cell bank able to match agreat number of people from diverse ethnic groups seems daunting at the present timebecause of the inefficiency of ES cell production and the limited availability of specificallydonated human blastocysts. Although the efficiency of successful human ES cell linecreation has been reported to be 43%, this high rate is probably achieved in only a few,experienced laboratories.25

Blood-based adult stem cells

The answer to this predicament might come from the discovery that adult-derivedstem cells are thought to maintain a capacity to differentiate into tissues different fromtheir tissue of origin.19,26 This has been controversial but also very illuminating. Thepossibility of easily accessible stem cells from non-controversial sources with a widerange of tissue capabilities immediately vaulted the adult stem cell field into theforefront of new therapies. Of all the new possible sources of stem cells available, themost useful ones would have to have a potentiality similar to that of ES cells and a strongcapacity to proliferate, or else be easy to collect in large numbers. For all tissue-specificstem cells, these are major hurdles to overcome. Although a clear winner is not yetapparent, some strong candidates are emerging. From these, bone-marrow-derivedstem cells or umbilical-cord-derived stem cells are strong candidates. The bonemarrow consists of two main cell types, mesenchymal and haematopoietic, with theformer currently showing a strong propensity for proliferation and multi-lineagedifferentiation.27–30 Furthermore, the haematopoietic stem cell is also showing promiseas a multipotential stem cell.31,32

With the promise of mesenchymal cells and blood cells as potential alternatives toES cells in some clinical situations, umbilical cord blood and peripheral blood have bothcome under consideration as a source of bone-marrow-like stem cells due to their ease


of collection compared to bone marrow. Umbilical cord blood has already provenextremely useful as a source of haematopoietic stem cells for bone marrowtransplantations.33,34

Umbilical cord blood haematopoietic stem cell transplantation

Umbilical cord blood was first used for a successful bone marrow transplant in 1988 in apatient with Fanconi’s anaemia.35 The majority of haematopoietic stem cells are foundin the fetal circulation and, therefore, in the placenta and umbilical cord. Thehaematopoietic stem cells migrate to the bone marrow within hours after delivery,where they take up residence and provide a life-long supply of stem cells andprogenitors of all the blood-forming elements, including erythrocytes, leucocytes andplatelets. Stem cells remain in the placenta and umbilical cord post-delivery and onaverage 120 ml of blood can be collected with no risk to either the mother or the baby.Furthermore, this rich source of stem cells is usually discarded.

Umbilical cord blood has more haematopoietic stem cells per volume thanperipheral blood or bone marrow. In addition, umbilical cord blood seems moretolerant of HLA mismatches, with less graft versus host disease.36 Non-invasivecollection37 is another major advantage over bone marrow. The number of umbilicalcord blood leucocytes per kilogram of recipient weight required to obtain a successfultransplant is reported to be between 15 and 50 million. However, the number ofhaematopoietic stem cells from a single cord is usually sufficient to engraft only childrenand small adults. We estimate that 25% of the samples stored with the Toronto CordBlood programme have sufficient numbers of cells to merit use for adulttransplantation.38 As a result of a combination of improved collection techniques andmore efficient cell processing and cryopreservation methods, the yield of stem cells isrising and might lead to successful engraftment of adults as well as children. For the year2001, we collected 2700 samples containing an average of one billion (109) leucocytesper single cord blood sample. The leucocytes contained an average of 0.54% CD34Ccells or 5.3!106 CD34Ccells pre sample. The range of cells per sample varies widely,with some samples containing as few as 100 000 CD34Ccells and others containingover 107 CD34Ccells (Figure 1). The variation observed is partly dependent on

Individual Patient Samples

CD34+ cells/sample

0

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(x m

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30

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Figure 1. Individual umbilical cord blood samples were tested for CD34Ccells by flow cytometry. CD34C

content ranged from 100 000 per sample to over 30 million CD34Ccells, with the average being 5.3 million

cells/sample.


the volume of blood collected. As expected, the collected cells are a perfect HLAmatch for the donor and an excellent match for siblings or other immediate familymembers.

Many cord blood transplantations have been performed to treat patients withmalignant and non-malignant diseases.39 Most of these have been from sibling donorswith partial or complete HLA matching. The incidence of acute graft-versus-hostdisease (GVHD) has been lower than the expected rate in a paediatric populationreceiving transplants of bone marrow. Interestingly, acute GVHD has not been linked tothe degree of HLA matching, suggesting that the use of cord blood itself is responsiblefor the reduced risk.40,41 As a result of tolerance of cord blood transplants to moreHLA mismatches, fewer cord blood stem cell donors should be required to meetworldwide needs than with bone marrow. In addition, in contrast to bone marrowtransplants in which general anaesthesia and surgical transfer of the donor marrow tothe recipient is needed, umbilical cord blood stem cell transplantation involves simpleintravenous infusion of the haematopoietic stem cells, which find their way to the bonemarrow for engraftment.

Haematopoietic stem cells derived from umbilical cord blood are easy to obtain andrelatively inexpensive to collect and store, as discussed in detail in Chapter 11. Thisallows for individuals to store their samples for possible future use even if there is noimmediate indication that stem cell therapy will be required. Public banks can be set up tostore readily available HLA-matched samples that could be retrieved within hours, notdays or weeks as required for bone marrow samples. In addition to the haematologicaldisease indications that normally require a bone marrow transplant, the availability ofHLA-matched cells make it feasible to use haematopoietic stem cells derived fromumbilical cord blood for indirect indications such as allowing more aggressive treatmentsof non-haematological cancers or to prevent or cure metastases. Rodriguez-Galindo et al(2003) used autologous bone marrow transplantation to successfully treat retinoblas-toma patients with bone metastases.42 Although successful, the risk of not removing allpossible cancercells during rounds of chemotherapy before bone marrow harvest alwaysremains a possibility. In a similar retinoblastoma case, a patient who banked cord blood inour programme utilized the sample for transfusion after chemotherapy. In light of the riskof metastatic disease, the availability and use of the stored cord blood sample collected atbirth was preferable to autologous bone marrow.

HAEMATOPOIETIC STEM CELL EXPANSION

Successful transplants and cell therapies are dependent on the speed at which thedonor cells contribute to the recovery of tissue function, and this in turn dependson cell numbers. Cells derived from the umbilical cord will always be in limitedsupply. To circumvent this problem, improvements in collection, processing andstorage of samples have reduced cell loses, which can be up to 20% during theprocessing of the blood to remove red blood cells and to reduce the volume. Usingcells from two different but related samples has occurred but is not practical in mostcases.43 Others, including our laboratory, have attempted haematopoietic stem cellexpansion.

Experimentally, the main hurdle to overcome in stem cell expansion is themaintenance of the self-renewing stem cell population during induced proliferationthus reducing the loss of self-renewing stem cells that occurs. Attempts to stimulate


haematopoietic stem cells to proliferate generally leads to differentiationsince growth factors have both mitogenic and differentiation properties.44 Thenormal fate of stem cells in vivo is to divide, producing one stem cell and onedifferentiating cell thereby maintaining a constant number of stem cells.45,46

Expansion is dependent on triggering proliferation without inducing the internaldifferentiation programme.

Although many laboratories have reported haematopoietic stem cell expansion,most of the reported successes are based on in vitro results, which do not realisticallymeasure engraftment potential of the cells.47 There are two types of repopulatinghaematopoietic cell: progenitor cells, which have limited renewal capacity, and stemcells, which have a much greater capacity for self-renewal.48,49 Most cytokine-supplemented growth conditions promote the differentiation of stem cells intoprogenitor cells and then eventually into mature cells. Colony-forming unit (CFU)assays tend to measure progenitor cells indirectly by determining the number ofmonopotential or bipotential cells in the input culture. True stem cells aremultipotential and capable of giving rise to all mature cell types. Long-term cell-initiating cultures (LTC-IC) measure the ability of cells to self-renew over 6 weeks, aproperty of stem cells, but do not measure the ability of cell to home to the bonemarrow.50,51 Even conditions that can increase the number of stem cells as measured bythe LTC-IC might not result in haematopoietic stem cell homing to the bone marrow,which is critical for transplant success.

Different growth factor conditions result in different expansion rates (Figure 2).Interleukin (IL)-3, for example, is a well-known cytokine capable of inducingproliferation in haematopoietic cells.52 Fgf-2 and Fgf-4 are important during embryodevelopment for the proliferation of cells. For example, Fgf-4 is responsiblefor elongation of the limb during development.53 Stem cell factor is also a welldocumented factor that is responsible for maintaining stem cells and reducing their rate

Day6 Day6 Day15 Day15CM+FGF4 1.3 1.8 1.6 5CM+FGF4+SCF 1.3 1.3 4 10CM+FGF4+IL3 2.2 3 20.8 75

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CD34+ CD45+ CD45+CD34+

Figure 2. Lineage negative blood cells were grown in conditioned medium (CM) with FGF4 plus stem cell

factor (SCF) or interleukin (IL)-3 for 6 or 15 days. Cells were assayed for CD45 and CD34 by flow cytometry.

At 6 days there is some cell proliferation with almost all cells maintaining CD34l; at day 15 there has been a lot

of cell proliferation, with most cells differentiating and losing the stem cell marker CD34.


of differentiation.54 We selected cells using a negative selection column, which allowedus to remove all mature cells and retain stem and progenitor cells (lineage minus cells).These retained cells were placed into cultures using human fetal fibroblasts as a feederlayer, IMDM with 10% serum and supplemented with growth factors, resulting inexpansion of the lineage minus cells. It has been reported by others that feeder cellsprovide a substrate similar to that which the haematopoietic stem cells are exposed toin the bone marrow microenvironment and thus aid in stem cell maintenance andproliferation.55 We tested this by substituting the feeder layer for medium conditionedfor 3 days on the feeder cells. The conditioned medium (CM) was used with the samegrowth factors and achieved similar results, suggesting that feeder cells act by secretinggrowth factors, and that cell–cell contact is not required. Expansion was dependent onthe factors used as illustrated in Figure 2. Under all conditions tested the stem/progenitor cells eventually differentiated. At day 6, proliferation rates were similar forall conditions, with IL-3-supplemented conditions having a slight advantage. Mostimportantly, the rate of stem cell expansion as measured by CD34 (progenitor cellmarker) was similar to that measured by CD45 (pan-haematopoietic marker),indicating that at day 6 the cells had not differentiated. By day 15 the composition ofthe cells had changed and the effects of the different supplementation regimes wasmore noticeable. Many of the cells had differentiated as the number of CD45Ccellexceeded that of the CD34Ccells. This was expected, as the progenitor cells in thepopulation will differentiate. Inevitably, the cultures will be depleted of all stem cells.The key is finding the right conditions and time that will lead to stem cell expansionbefore differentiation and extinction of the stem cell population.

MESENCHYMAL STEM CELLS

Bone marrow mesenchymal cells are distinct from haematopoietic cells and mainlydistinguished by being CD45K. Mature bone marrow mesenchymal cells are a mixedpopulation of cells that are capable of supporting haematopoiesis and differentiatinginto endothelial, bone, muscle and neural cells. Different reports suggest that a singlemesenchymal progenitor cell, or possibly two cells, accounts for all reported lineagesthat have been produced from bone marrow mesenchymal cells in vitro. The recentdemonstrations of bone marrow cells (mesenchymal progenitor cells) giving rise toother tissues such as muscle56–58, liver59,60, neural61,62 or multiple cell types dependingon their experimental environment, support the potential of tissue-specific stem cells(also called adult stem cells) for cellular and tissue therapy.19 The ability of a clonalpopulation of murine mesenchymal progenitor cells to contribute to multiple tissuetypes has recently been illustrated.26 Single mesenchymal progenitor cells (multi-potential adult progenitor cells or MAPC) from rats have the ability to undergo120 divisions and give rise to cell types of all three germ layers: endoderm, mesodermand ectoderm. These cells seem to require LIF (Leukemia Inhibitory Factor) for theirsurvival, and also express the ES cell markers OCT-4 and Rex-1 and SSEA-126; humanbone marrow stromal cells show similar properties. Isolated human bone marrowmesenchymal cells were induced to undergo neuronal differentiation in vitro,expressing neuronal specific enolase (NSE) and tubulin. These cells followed a normaldifferentiation pathway, initially expressing nestin, a marker of neuronal precursors,within 5 hours of induction, followed by a decrease in nestin and an increase in NSElevels.63 In another study, haematopoietic stem cells were purified and clonal


populations were examined to determine their self-renewal and differentiationpotential. Long-term repopulation of irradiated hosts was used to show that thesecells migrate to the bone marrow but can also differentiate into epithelial cells of theliver, lung, gastrointestinal tract and skin.19

MAPC cells are obtained more easily from younger donors. This is perhaps becauseof the depletion of self-renewing stem cells in our tissues as we age, or because of theloss of multipotential capacity of these cells over time. The fact that HLA mismatchesare better tolerated with haematopoietic stem cells derived from umbilical cord blood,resulting in less GVHD, suggests that the cells derived from umbilical cord blood aremore adaptable and have not fully differentiated. This flexibility could go beyond HLAmatching and extend into differentiation capabilities. If this is in fact the case, then amultipotential blood stem cell or an MAPC-like cell might be more abundant inumbilical cord blood than in the bone marrow of older patients.

Does umbilical cord blood contain mesenchymal-like stem cells? Or do the bloodcells demonstrate a multipotential capacity? Our data suggest that we can respond ‘yes’to both of these questions, although a definitive answer is not yet available. The abilityof umbilical-cord-derived stem cells to produce non-haematopoietic cells has not beenextensively documented. Three groups have reported the isolation of a mesenchymalprogenitor cell from umbilical cord blood.64–66 These studies conclude that the abilityof umbilical cord blood to produce non-blood cells is due to the presence ofmesenchymal-like cells. Mesenchymal cells were isolated from the Ficoll layer ofumbilical cord blood and formed an adherent population that allowed their separationfrom haematopoietic progenitor cells by differential adherence selection.66 Mesench-ymal cells from umbilical cord blood were CD45K, CD34K and did not express theendothelial marker, CD31; this is similar to mesenchymal cells in bone marrow and infirst-trimester fetal blood.67 After 3 weeks of growth in serum-containing medium, 25%of the cultures had mesenchymal cell properties as defined by morphology and FACSanalysis. Growth in osteoblast differentiation medium or adipocyte medium resulted inpositive osteoblast-like or lipid-containing cells. To determine whether the cellsisolated from umbilical cord blood are truly multipotential Lee et al64 first depletedmature blood cells from the starting cultures prior to culture. They then plated 30 cellsper well and cultured them through several passages before testing their potential.Although they were unable to grow single cells, the low number of cells in the startingcultures and the wide range of mature cell types generated from single cultures isstrongly suggestive of multipotentiality.

A comparison of bone-marrow-derived and umbilical-cord-derived mesenchymalcells revealed that bone marrow contains more mesenchymal progenitor cells. Theyield of mesenchymal stem cells from umbilical cord blood is very low—even lowerthan in first-trimester blood—which might be why the cells are difficult to detect.68 Wehave managed to isolate umbilical-cord-blood mesenchymal cells that morphologicallyresemble the bone-marrow-derived cells. These umbilical cord blood cells will adhereto plastic in medium containing 20% serum. We obtained 1000–5000 cells per umbilicalcord sample. The cells tend to divide once or twice over a 2–3-week period andcontinuous cultures are difficult to establish. These results are similar to those ofGoodwin et al69, who also report isolating an adherent population of cells fromumbilical cord blood that grow slowly but are capable of forming osteoblasts,adipocytes and cells expressing neural markers. Although they do not report explicitlyon the CD45 pattern of their starting population, the cells are CD45K after culturing.In our experiments, the starting population is CD45C, as was the case in theexperiments by Mareschi et al65 but the cells lose CD45 during the in vitro growth


phase. In our experience, these cells have limited use, due to the low numbers of cellsgenerated from an individual umbilical cord blood sample. However, this is likely tochange as we discover growth conditions that will promote their proliferation. It ismost likely that mesenchymal cells derived from umbilical cord blood face similarhurdles to proliferation as those facing the proliferation of MAPC cells.

MULTIPOTENTIAL UMBILICAL CORD BLOOD CELLS

We must proceed with caution in determining a possible mechanism to explain themultipotential properties observed with adult cells. It has not been determinedwhether adult stem cells: (i) can be reprogrammed; (ii) are rare stem cells similar to EScells; or (iii) fuse with the host cells and stimulate repair of injured or diseased tissues.The appearance of cell-specific markers during in vitro studies does not guaranteefunctionality, which is the important end point in these studies. To use these cells safelyfor tissue therapy, the mechanism(s) of cell differentiation must be elucidated. To date,the studies reported contradict each other, and also contradict what is observed duringendogenous repair of the human body.70,71

TRANSDIFFERENTIATION STUDIES

In vitro studies are carried out with putative stem cells without accessory cells and,therefore, rule out the possibility of fusion as the mechanism for the observed plasticity.But the majority of positive results reported have been dependent on using detectionmethods for differentiation that are based on gene and protein expression rather thanfunctional studies.72–74 The expression of single markers as determined by polymerasechain reaction (PCR) or immunocytochemistry might not tell us the full differentiation

Figure 3. Umbilical cord blood cells were grown in medium with 10% serum and tested for osteoclast cells by

absorption of a calcium carbonate substrate. The cells required the addition of GM-CSF to obtain functional

osteoclast cells.


potential of the cell in question but does provide preliminary evidence and suggests thatfurther investigation is merited. Furthermore, transdifferentiation might not need tocross germ layer boundaries to be clinically relevant. Differentiation into different butrelated cell types could still provide us with clinically relevant populations. Osteoclastsare of monocyte origin and function in bone remodelling.75 Differentiation of umbilicalcord blood monocytes into osteoclasts has been successful. Growth of monocytes inserum containing medium supplemented with GM-CSF caused the differentiation of thecells into TRAP-positive cells capable of digesting a calcium substrate used to identifyfunctional osteoclasts in vitro (Figure 3). Similarly, neural microglial cells are alsomonocyte derivatives and thus might be easier to derive from blood than other neuralcells. Endothelial cells share a common precursor with blood cells. The hemangioblastis generally considered an embryonic cell but might still persist in the fetal and placentalcirculation.76 There have been reports of the isolation of endothelial cells fromumbilical cord blood.77 The exact lineage of the endothelial cells has not been fullydetermined. It is possible that they might result from the endothelial cells lining thevessels within the umbilical cord and not the blood. If the origin of these cells is fromthe blood vessels then saving the cord as well as the blood might be necessary.

FUSION STUDIES

Fusion is another mechanism that might explain the multipotential of blood cells andprovide for a mechanism that would increase the potential use of umbilical cord bloodcells. The ability of cells to fuse with like cells occurs naturally in the body. Muscle cellmaturation is dependent on the fusion of individual myoblasts.78 In the liver,hepatocytes also fuse, resulting in polyploidy.79 Recent evidence indicates that donorcells might fuse with surrounding cells and adopt their fate, suggesting that although thestem cell is contributing to the repair of the tissue, the mechanism might be by fusionand not transdifferentiation.80–82 Although conflicting results have been reported withumbilical cord blood cells contributing to liver repair by fusion, the datasuggest that stem cells are not required and fusion would be the most likelyexplanation.31,73 If this proves correct, then the limitation facing cord blood use due tolow numbers of available stem cells is not relevant here. With the average cord bloodsample yielding one billion mature cells, the probability of there being enough cells forfusion-based cell therapy remains high.

The liver provides us with an ideal model for untangling the different mechanismsthat result in tissue repair of non-haematopoietic tissue from umbilical cord blood. Theliver is easy to access surgically and the animals can survive long enough after partialhepatectomy to allow repair of the liver to occur. Liver is also unique in that it is capableof repair through the division of hepatocytes (progenitor/mature cell) or from the ovalcell (stem cell) population.83,84 Published reports suggest that blood cells can fuse withhepatocytes and contribute to liver tissue repair.85 It is less clear whether thecontributing blood cell is a mature cell or a stem cell, and whether or not fusion is thesole mechanism. As there have been reports of tissue-specific stem cells contributing tothe repair of unrelated tissues, it is possible that the mechanism of fusion,transdifferentiation or the existence of a universal stem cell is common to all ofthese reports. There is strong evidence that bone marrow cells can contribute to liverrepair in humans. A retrospective study of bone marrow transplant patients in whom


there was discordance of donor–recipient gender clearly showed that bone marrowcells contributed to functional liver cells within the recipient.59,60

SUMMARY

Blood cells, including those from umbilical cord, exhibit the potential to contribute tothe repair of damaged organs, including liver, endothelial and possibly neural and musclecells. Although the umbilical cord stem cells do not demonstrate the same variedpotential as ES cells, they might be effective for some clinical situations. Currently, lowcell numbers and the inability to deliver the cells to the targeted area hamper theirwidespread use. The development of tissue-specific stem cells for cell and tissuetherapy is still in its infancy but holds great promise for the future.

Research agenda

† growth factor supplement during stem cell growth in vitro† identification of the multipotential stem cell in umbilical cord blood† develop mouse models of human diseases for in vivo testing of umbilical-cord-

derived cell therapies

Practice points

† umbilical cord blood cells are an effective haematopoietic stem cell source forbone marrow transplants

† although umbilical cord stem cells are used mainly for paediatric transplants,successful adult transplants have occurred

† umbilical cord blood stem cells are easily cryopreserved, and survive for manyyears, which allows for the establishment of stem cell banks

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