The umbilical cord: a rich and ethical stem cell source to advance …

Cell Prolif. 2011 Apr; 44(Suppl 1): 6069.

1 CTILYON, Cell Therapy Research Institute, Parc Technologique de Lyon St Priest, St PriestLyon, France

1 CTILYON, Cell Therapy Research Institute, Parc Technologique de Lyon St Priest, St PriestLyon, France

1 CTILYON, Cell Therapy Research Institute, Parc Technologique de Lyon St Priest, St PriestLyon, France

Received 2010 Jun 24; Accepted 2010 Aug 11.

Science and medicine place a lot of hope in the development of stem cell research and regenerative medicine. This review will define the concept of regenerative medicine and focus on an abundant stem cell source neonatal tissues such as the umbilical cord. Umbilical cord blood has been used clinically for over 20years as a cell source for haematopoietic stem cell transplantation. Beyond this, cord blood and umbilical cordderived stem cells have demonstrated potential for pluripotent lineage differentiation (liver, pancreatic, neural tissues and more) in vitro and in vivo. This promising research has opened up a new era for utilization of neonatal stem cells, now used beyond haematology in clinical trials for autoimmune disorders, cerebral palsy or type I diabetes.

Stem cells; never in the history of science and medicine have two words sparked off so much interest, passion, controversy and hope from the scientific, medical, public, ethical, religious, political and commercial communities. It is, however, important to state clearly that despite many significant clinical achievements and great promises, stem cells are not the sole means to cure all diseases. Biomedical research and future treatments will always rely on innovation in medicine, surgery, technology and/or pharmaceutical developments.

This review will outline concepts surrounding stem cell applications and regenerative medicine and focus particularly on a fascinating and abundant stem cell source the umbilical cord. This tissue physiologically supports development of the child throughout foetal life until birth, and can further be used for biomedical research and clinical applications.

The advancement of science, medicine and surgery has helped mankind improve global health, albeit with significant disparities in accessing healthcare worldwide between developed and emerging countries, but many definitions have been proposed for the term regenerative medicine (1, 2, 3). Kaiser, a health economist forecasting future medical technologies, first presented this concept in 1992 as an attempt to alleviate chronic diseases and restore damaged and failing organs (4).

With the development of immunosuppressive regimens, transplantation medicine and surgery in the 20th century and now the 21st, have enabled treatment of patients who would have had no therapeutic alternatives. However, shortage of donor organs increased significantly with clinical demand.

Taking liver as an example, it is estimated that 70% of patients awaiting liver transplantation in European Union countries will never find a donor. This persistent shortage of liver donors has led to mortality rate of 20% per year from the waiting list (5).

Our modern lifestyles have also increased prevalence of diabetes (type I and II) and cardiovascular diseases, which both cause major complications (stroke, kidney failure and more), and in the USA alone account for annual health care costs as high as 174billion dollars and 475billion dollars, respectively (source: USA National Institute of Health).

These challenges represent opportunities for the field of regenerative medicine. This aims to gather different scientific specialties and technologies to restore impaired functions in tissues and organs that have been damaged by illnesses, accidents or even by treatments.

Innovation and research in nanotechnologies, biomaterials, tissue engineering, bioimaging, cells and stem cells are key to the advance of regenerative medicine, as demonstrated with recent case studies and clinical trials. For instance, in 2006, Atala and colleagues isolated cells from patients with bladder dysfunction and cultured each patients own (autologous) cells on bioscaffolds in the shape of a bladder, in the laboratory. These artificially engineered bladders were later successfully reimplanted into the patients and restored their function (6). More recently, Macchiani and colleagues of an international consortium transplanted a young adult in Spain with a tissue engineered trachea segment. The donor trachea was made acellular and seeded with the recipients own epithelial cells and mesenchymal stem cellderived chondrocytes that had been cultured in vitro (7, 8). This technique was repeated for a child using a longer trachea segment in 2010 in the United Kingdom. So far, no complications have been reported as donor tracheas were decellularized and further reconstructed using the patients own cells. No rejection nor immune complication has been reported. At the time of writing, both patients are leading a normal life without immunosuppression (18months and 5months posttransplantation, respectively).

Such exciting and safe clinical cases of modern regenerative medicine illustrate the need for interdisciplinary research and understanding the full potential of stem cells for clinical applications.

Stem cells are defined by their capacity to divide and produce (at least one) identical stem cell (selfrenewal) and for one to undergo lineage differentiation (9). Depending on potency of stem cells to produce one or more lineages, they can be identified as totipotent (for example the zygote, the only mammalian cell capable of producing all cells and tissues of an organism), pluripotent (with capacity to produce cells and tissues from all three germ layers ectoderm, mesoderm and endoderm), multipotent (capacity to produce more than one cell lineage) or unipotent (differentiation into a single cell phenotype).

Early in the 1900s, Maximow was the first to propose that lymphocytes acted as common stem cells and migrated through tissues to form blood circulation components (10), but the 1960s shaped the true beginning of stem cell research as we know it today. Research by Till and McCulloch (11) and by Goodman and Hodgson (12) demonstrated in mice, that the bone marrow hosted stem cells, from which clonogenic precursors could be derived and could restore haematopoiesis in irradiated animals. This work simultaneously evolved with the advent of human stem cell transplantation for bone marrow replacement (13). At the same time, research by Edwards and colleagues generated the first embryonic stem cell lines in rabbits (14), which much later advanced into the development of the first human embryonic stem cell line (15). Stem cells today can also be categorized according to the source of tissues from which they originate.

Human embryonic stem cells (ESC) are derived in vitro from the blastocyst of an embryo usually left over from in vitro fertilization. ESC are cell lines derived from the embryoblast of early embryo at the blastocyst stage. They proliferate in vitro while maintaining an undifferentiated state, and are capable of differentiating into many specialized somatic cell types under appropriate conditions (pluripotency). Much fascination and controversy has fuelled the world of biomedical research since derivation of the first human embryonic stem cell lines, as it induces destruction of a human embryo. However, beyond ethical objections raised by research on human ESCs, significant technical hurdles have slowed their progress towards clinical application, not least their immunogenic status, spontaneous formation of teratocarcinomas upon transplantation and genetic/genomic instability in cell culture systems during scaleup (16).

Adult or somatic stem cells can be isolated from specific adult human tissues (brain, skin, gut, bone marrow, fat, cornea and more). They have limited ability to regenerate damaged tissues physiologically. Although their differentiation potency is considered to be less than ESC by some scientists, their isolation, characterization and translation to preclinical and clinical studies have increased during the past two decades, not least in the field of haemato/immunotherapies, but also recently for certain cardiovascular indications (17), wound healing (18, 19), corneal repair (20) or even although less advanced, for multiple sclerosis (21). Beyond tissuespecific stem cells, mesenchymal stem cells (MSCs) were first characterized as a specific bone marrowderived fibroblastlike adherent cell population with potential and capacity to support haematopoiesis (22, 23). Further studies demonstrated their potential to differentiate initially into three specific lineages: osteocytic, chrondrocytic and adipose lineages and later on into many endodermal, mesodermal and ectodermal tissues (24, 25).

Different adult tissues have been proposed as sources for MSCs: bone marrow, adipose tissues, synovium, dental pulp and more. Clonogenic assays and putative markers have also been proposed for MSCs, the biology of which is becoming better understood and standardized (26).

The recent discovery of induced pluripotent stem cells (IPS), by the initial work of Yamanaka and colleagues first in mice then in humans, has circumvented to a certain extent some ethical and scientific limitations of ESC research (27). This technique consists of using somatic terminally differentiated cells and inducing expression of a number of genes therein, to produce stable lines of embryoniclike pluripotent stem cells. This technique offers the interesting possibility of creating patientspecific stem cell lines for research, and perhaps oneday, diagnostic applications without the controversial use/destruction of human embryos. However, its significance for relevant clinical applications remains unknown as yet, mostly because of the low efficiency of such induced gene expression. New techniques are being investigated to generate IPS cells with minimal or no exogenous genetic modifications (28).

Our group previously proposed a distinct category of somatic stem cells called neonatal stem cells, derived from various biological tissues often considered as biological waste after birth, rather than biological resources, namely amniotic fluid, placenta, umbilical cord and cord blood (29, 30). With over 135million births per year worldwide (source: USA Central Intelligence Agency Factbook 2009), neonatal tissues are objectively the largest and most genetically diverse stem cell source that can be accessed in a noninvasive, rapid and costeffective manner during and after birth. This review will particularly focus on the umbilical cord as a stem cell source for biomedical research and clinical applications.

From the third week of development, the human embryo becomes attached via a connecting stalk to the forming placenta. At week 5, a primitive umbilical cord is formed in the shape of an umbilical ring. At week 10, after development of the gastrointestinal tract in the foetus, the umbilicus appears as a hernia linking into the umbilical cord.

The umbilical cord is covered by an amniotic epithelium which protects a gelatinous and elastic matrix made of mucopolysaccharides (mostly hyaluronic acid and chondroitin sulphate) called Whartons jelly named after Dr Thomas Wharton who first described it in 1656 (31). The amnion and Whartons jelly protect three blood vessels that are crucial for embryonic and foetal development. One large umbilical vessel supplies the developing foetus with placental blood, rich in nutrients and oxygen, and in the last trimester with important antibodies provided by the mother. Two smaller umbilical vessels return from the foetus the blood with carbon dioxide, wastes and other toxins.

The umbilical cord can provide stem cells from the blood running in the umbilical vessels, walls surrounding the vessels and from the Whartons jelly.

Cord blood can be collected at birth using a sterile collection kit consisting of an anticoagulant (usually citrate or heparin)containing collection bag connected to one or several collecting needle(s). Cord blood samples can be collected in utero, after the birth of the child and before delivery of the placenta or ex utero, from normal deliveries and caesarean sections with no pain for the mother or the child ().

Umbilical cord Whartons jelly as a source of mesenchymal stem cells. (a) Sagittal section of a 1cm diameter umbilical cord with Whartons jelly (WJ) surrounding two arteries (A) and one vein (V). (b) 10mm3 biopsy pieces of Whartons jelly. (c) Whartons jelly piece (WJ) in serumfree culture growingout mesenchymal stem cells. (d) Mesenchymal stem cells from umbilical cord at 80% confluence in serumfree culture.

In a recent study, our group demonstrated that cord blood stem cells and other cell populations, in general, were influenced by obstetric history and other maternal factors (30). Cord blood units are usually transferred to a laboratory where they undergo cell separation to extract the buffy coat and/or cell preparations enriched with stem cells. Different techniques exist to extract cells from cord blood, such as: centrifugal elutriation, rouleaux formation, starchbased methods, and densitygradient methods, among others (32, 33, 34) ().

Historically, umbilical cord blood has been known to contain haematopoietic stem/progenitor cells with that have the ability to produce clonogenic progeny. In 1974, Knuddtzon (35) was the first to confirm presence of cells with haematopoietic clonogenic potential in cord blood in vitro. Broxmeyer and colleagues published in 1989 a report confirming presence of haematopoietic stem cells in cord blood (36). Further studies verified their clonogenic potential, selfrenewal property and capacity to be expanded in vitro (37, 38, 39, 40, 41).

Our research group and others spent many years analysing the different cell groups present in cord blood to distinguish between stem cell and progenitor cell phenotypes, and later on between haematopoietic and nonhaematopoietic cell groups (40, 42). In 2004, our team reported for the first time, the discovery of nonhaematopoietic pluripotent embryoniclike stem cells from cord blood, named cord blood embryoniclike stem cells (CBEs). Further investigation of these cells demonstrated that they could be harvested from fresh and cryopreserved units and had expansion and differentiation potential into neural, hepatobiliary and pancreaticlike precursors (43, 44, 45, 46, 47, 48, 49, 50). This groundbreaking discovery, albeit challenged at the time, has since been confirmed by several other groups (51, 52, 53, 54, 55).

Further to this, our group and others have also been able to identify and isolate multipotent MSCs from cord blood with more restricted differentiation potential and significant variability between samples (unpublished observations; 56, 57, 58).

These studies have demonstrated that cord blood has potential beyond haematopoietic differentiation and could be considered for further regenerative medicine research (59).

Several groups have recently reported the possibility of deriving MSCs, not only from cord blood but also from the umbilical cord matrix Whartons jelly. McElreavey and colleagues first reported in 1991 the possibility of isolating fibroblastlike cells with population growth potential from the Whartons jelly (60). Several techniques have been reported to dissect Whartons jelly mechanically and/or digest it enzymatically to culture homogeneous MSC populations.

The French Academy of Medicine presented a report in January 2010 in which they considered that research on umbilical cord stem cells was extremely promising and could provide useful new tools for treatment of several diseases. A single piece of 510mm3 Whartons jelly has the potential to yield as many as 1billion MSCs in 30days (Degoul O., Jurga M., Forraz N. and McGuckin C.P. unpublished personal data). With the average umbilical cord measuring 50cm, one could predict that this source of MSCs will become more and more clinically relevant as research advances (). Umbilical cord Whartons jellyderived MSCs are increasingly being considered as more robust than those from cord blood itself and, by nature, they are less invasive than those from the bone marrow (61). Several studies have shown that umbilical cordderived MSCs can be differentiated into bone (62, 63), skin (64), endothelium (65), hepatocyte (66, 67) and neural lineages (68) to name but a few. The immunomodulatory properties of umbilical cord MSCs were shown to be similar to bone marrowderived MSCs (69). The potential of this stem cell source is therefore enormous for regenerative medicine applications.

Umbilical cord blood collection and processing. (a) Cord blood is collected after birth from the umbilical vein into (b) citratebased anticoagulantcontaining blood collection bag. (c) Sepax device, enabling closed system cord blood processing in approximately 20min.

Follow this link:

The umbilical cord: a rich and ethical stem cell source to advance ...