Placenta—an alternative source of stem cells

Placenta—an alternative source of stem cells

Toxicology and Applied Pharmacology 207 (2005) S544 – S549 Review Placenta—an alternative source of stem cells Tiina M...

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Toxicology and Applied Pharmacology 207 (2005) S544 – S549


Placenta—an alternative source of stem cells Tiina Matikainen a, Jarmo Laine b,* a

Program of Developmental and Reproductive Biology, Biomedicum Helsinki and Hospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland b Stem Cell and Transplantation Services, Finnish Red Cross Blood Service, Kivihaantie 7, FIN 00310, Helsinki, Finland Received 15 July 2004; revised 20 January 2005; accepted 31 January 2005 Available online 28 June 2005

Abstract The two most promising practical applications of human stem cells are cellular replacement therapies in human disease and toxicological screening of candidate drug molecules. Both require a source of human stem cells that can be isolated, purified, expanded in number and differentiated into the cell type of choice in a controlled manner. Currently, uses of both embryonic and adult stem cells are investigated. While embryonic stem cells are pluripotent and can differentiate into any specialised cell type, their use requires establishment of embryonic stem cell lines using the inner cell mass of an early pre-implantation embryo. As the blastocyst is destroyed during the process, ethical issues need to be carefully considered. The use of embryonic stem cells is also limited by the difficulties in growing large numbers of the cells without inducing spontaneous differentiation, and the problems in controlling directed differentiation of the cells. The use of adult stem cells, typically derived from bone marrow, but also from other tissues, is ethically non-controversial but their differentiation potential is more limited than that of the embryonic stem cells. Since human cord blood, umbilical cord, placenta and amnion are normally discarded at birth, they provide an easily accessible alternative source of stem cells. We review the potential and current status of the use of adult stem cells derived from the placenta or umbilical cord in therapeutic and toxicological applications. D 2005 Elsevier Inc. All rights reserved. Keywords: Stem cells; Placenta; Regenerative medicine; Toxicology

Contents Main text. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cord blood, umbilical cord, placenta and amniotic membranes/fluid regenerative medicine . . . . . . . . . . . . . . . . . . . . . . . . Cord blood and umbilical cord . . . . . . . . . . . . . . . . . . Placenta, amniotic membranes and fluid . . . . . . . . . . . . . Derivation of hepatocyte-like cells from human placenta . . . . Use and application of stem cells in toxicology . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Fax: +358 9 5801429. E-mail address: [email protected] (J. Laine).

After human embryonic stem cell lines were first described in 1998 (Thomson et al., 1998), there has been a dramatic increase in research into the biology and practical

0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.01.039

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application of stem cells. As stem cells are able to renew themselves and to differentiate into a number of specialised cell types, they could conceivably become widely used in cellular replacement therapies in the treatment of a large number of common human diseases including diabetes, neurodegenerative disorders, ischaemic diseases (myocardial and brain infarct), spinal cord injury, etc. Other promising applications of stem cells have been recognised in toxicology, where their use is thought to facilitate more rapid testing of candidate drug molecules in a context resembling normal cellular developmental programs or in stem cell derived specialised cell models of end-organ toxicity (e.g., cardiotoxicity, hepatotoxicity and reproductive toxicity) (Davila et al., 2004; Gorba and Allsopp, 2003; Rolletschek et al., 2004). In order for the potential of stem cells to be realised, they must be available in high numbers, and they should be easy to isolate, purify, expand in number without induction of spontaneous differentiation and to differentiate into the cell type of choice. This requires an easily accessible plentiful source of stem cells. Stem cells can be classified into embryonic and adult stem cells. Embryonic stem (ES) cells are pluripotent and can give rise to all specialised cell types of the organism. Human ES cells are derived from the inner cell mass of an early preimplantation embryo-embryonic day 5 blastocyst. In this process, the cells of the inner cell mass are removed from the blastocyst and cultured in vitro in order to establish an ES cell line. The cell line remains pluripotent and can theoretically be induced to differentiate into any specialised cell type of choice (Thomson et al., 1998; Kehat et al., 2001; Lavon and Bevenisty, 2003; Reubinoff et al., 2000). The use of ES cells has raised significant ethical discussion since the early blastocyst is destroyed when inner cell mass is removed. Legislation and public acceptance regarding the use of EScells varies in different countries, with the U.S.A. having limited federal research funding to lines already established by August 2001. Legislation in most European (excluding Germany and Italy) and Asian countries remains permissive. The use of ES cells is further made difficult by poor success rate in establishing cell lines from the blastocysts. Currently, only circa 100 cell lines have been established world-wide. All cell lines are not well characterised and lack of availability remains a significant obstacle for research. Adult stem cells are rare cells (1: 107 –108 of all cells) thought to be present in all tissues and responsible for maintaining the specific tissue. The classic adult stem cell is the haematopoietic stem cell (HSC) which is capable of regenerating all haematopoietic cell lineages. HSCs have been used in clinical medicine since 1970s in bone marrow transplantation after cytotoxic therapies. Originally, the differentiation capacity of adult stem cells was thought to be limited to replenishing their specific progeny, but a large number of studies have in recent years demonstrated that their capabilities often extend to other somatic cell types as well. However, the full differentiation potential of adult


stem cells remains unclear. Although highly multipotent adult stem cells have been isolated at least from bone marrow, fat, brain, skeletal and cardiac muscle (Beltrami et al., 2003; Jiang et al., 2002; Pittenger et al., 1998; Wagers et al., 2002; Zhao et al., 2002; Zuk et al., 2002), there is no conclusive evidence that these cells are truly pluripotent. The use and research of adult stem cells is less controversial than that of ES cells since adult cells can be harvested from among self-renewing cells of volunteer donors (e.g., bone marrow). However, harvesting the cells remains invasive and although risks to the donor are quite small, they still exist. Since cord blood, umbilical cord, placenta and amniotic membranes and fluid are normally discarded after birth, they represent a non-controversial and highly available potential source of adult stem cells. Further, harvesting cells from these tissues is completely noninvasive and safe.

Cord blood, umbilical cord, placenta and amniotic membranes/fluid as sources of stem cells and their use in regenerative medicine Cord blood and umbilical cord Umbilical cord blood has been long recognised as an ample source of haematopoietic stem cells. Cord blood can be relatively easily collected from the umbilical vein of the otherwise discarded placenta after birth. There is currently no single specific marker which could be used to quantify or isolate the haematopoietic stem cells. Of the total nucleated cell number of circa 1– 2  109 which can often be obtained from a single cord, approximately 2– 3  106 will be positive for the most commonly used haematopoietic stem cell surface marker CD34. Cord blood is routinely used as an alternative for bone marrow transplantation in the treatment of haematopoietic malignancies (Barker and Wagner, 2003; Grewal et al., 2003). Several countries have established public cord blood banks, which collect cord blood after full-term pregnancies and store it in liquid nitrogen for possible future use in patient care. Currently, the world wide inventory of cord blood units in public banks is circa 150 000. When compared as a therapeutic option with allogeneic bone marrow/peripheral blood stem cell transplantation, platelet and neutrophil engraftment of cord blood transplants is slower and the patients longer at risk for infection, bleeding and early mortality. On the other hand, the use of cord blood is associated with a lower incidence of significant graft versus host disease than the use of bone marrow. Most recent reports suggest that the long term results of cord blood transplantation are equal to those obtained with bone marrow (Barker and Wagner, 2003; Grewal et al., 2003). However, the use of cord blood is still limited by the relatively small number of cells obtained from the umbilical vein and can usually be administered only to pediatric patients.


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Table 1 Reported differentiation potential of stem cells derived form cord blood, umbilical cord, amniotic membranes and placenta Cell source

Cord blood

Umbilical cord matrix

Amniotic epithelial cells


Differentiation potential

Hematopoietic lineages

Neurons Glia Adipocytes Osteoblasts

Neurons Chondrocytes Insulin-producing cells

Hematopoietic lineages

(Mitchell, 2004; Mitchell et al., 2003; Romanov et al., 2003)

(Bossolaco, 2004; Nikaido, 2004; Strom and Miki, 2003; Wei et al., 2003)

Neurons Glia Oligodendrocytes Astrocytes Adipocytes Osteoblasts Chondrocytes Hepatocyte-like cells (Buzanska et al., 2002; Goodwin et al., 2001; Lee et al., 2004; Sanchez-Ramos et al., 2001)

Since cord blood is readily available, a number of investigations have aimed to elucidate whether the stem cells it contains can be induced to differentiate into cells of other tissues beyond blood. Several studies have demonstrated that adult stem cells can be isolated and propagated from cord blood using a number of selection techniques and growth media (Buzanska et al., 2002; Goodwin et al., 2001; Lee et al., 2004; Sanchez-Ramos et al., 2001). The multipotent cells contained in the cord blood have in one investigation been shown to be able to differentiate into cells of all three germ layers, e.g., neural-, hepatocyte-, osteoblast-, adipocyte- and chondrocyte-like cells (Lee et al., 2004), and in a number of studies into neural-, osteoblast- and adipocyte-like cells (Table 1) (Buzanska et al., 2002; Goodwin et al., 2001; Sanchez-Ramos et al., 2001). Compared with the more conventional source of nonhematopoietic adult stem cells—bone marrow—there have thus far been only a limited number of reports describing results of pre-clinical or clinical investigations using stem cells of cord blood, amniotic or placental origin. Intense research focusing on alternative therapeutic indications is currently in progress (Table 2). In pre-clinical NOD-SCID-mouse models, the administration of cord blood-derived stem cells has been reported to lead to improved recovery from myocardial and hind-limb ischemia (Botta et al., 2004; Finney, 2004) secondary to

Hepatocyte-like cells Pancreatic a-, h-, y-cells Vascular endothelial cells Neurons Glia Oligodendrocytes Astrocytes (Alvarez-Silva et al., 2003; Strom and Miki, 2003)

improved perfusion and vascularisation of the site of injury. Promising results have also been obtained in a rat brain infarct model, in which intravenous administration of unselected cord blood nucleated cells resulted in improved recovery of motor function (Chen et al., 2001). In addition to cord blood, adult stem cells have been isolated and propagated from the umbilical cord matrix (Mitchell, 2004; Mitchell et al., 2003; Romanov et al., 2003). These cells have been reported to be able to differentiate into neural-, osteoblast- and adipocyte-like cells (Table 1). Placenta, amniotic membranes and fluid The formation of placenta (Zhou et al., 2003) is prerequisite for successful pregnancy. After fertilisation, implantation requires trophoblast differentiation, followed by rapid assembly of these embryonic cells into a functional placenta. Ectodermal stem cells termed cytotrophoblasts attach the placenta into the uterine circulation. As a result, the organ is ideally positioned to perform a wide variety of functions during pregnancy, including gas, nutrients and waste exchange. In a subset of placental chorionic villi, cytotrophoblasts begin to behave like tumour cells. They may leave the trophoblast basement membrane, where they are anchored in the placenta proper, and form aggregates that attach the conceptus to the uterine wall. Cytotropho-

Table 2 Current status of cord blood, placental and amniotic stem cells in tissue repair Cell source




Cord blood

Hematopoietic stem cell transplantation Limb ischaemia Myocardial ischaemia Brain infarction Liver failure Cornea repair Spinal cord injury repair Brain infarct Parkinson’s disease

Routine clinical Pre-clinical Pre-clinical Pre-clinical Pre-clinical Clinical Pre-clinical Pre-clinical Pre-clinical

Comparable to bone marrow (Barker and Wagner, 2003) Improved circulation (Finney, 2004) Improved recovery (Botta et al., 2004) Improved recovery (Chen et al., 2001) Unpublished Improved regeneration (Shimmura and Tsubota, 2002) Integration to site of injury (Sankar and Muthusamy, 2003) Integration to site of injury (Sakuragawa et al., 1997) Partial alleviation of symptoms (Kakishita et al., 2003)

Placenta Amniotic membrane

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blasts from these sites invade the interstitium of the decidua, eventually reaching the inner third of the myometrium (Zhou et al., 2003). The invasive character of the cytotrophoblasts should be kept in mind when clinical safety of placental derived stem cells and their applications is assessed. Placenta has an important role as a haematopoietic organ. During mouse embryogenesis early haematopoietic progenitor cells were found to be 2- to 4-fold more numerous in the placenta than in fetal liver or yolk sac, which have previously been established as sites of extramedullary haematopoiesis (Alvarez-Silva et al., 2003). These progenitors are in principle capable of forming all haematopoietic cell lineages of the adult animal. In addition to haematopoietic stem cells, placenta has been reported to contain a population of multipotent stem cells demonstrating some of the characteristics of pluripotent ES cells including expression of stem cell markers c-kit, Thy-1, OCT-4, SOX2, hTERT, SSEA1, SSEA3, SSEA4, TRA1-60 and TRA1-81. These cells resemble mesenchymal stem cells and can be induced to differentiate into hepatocyte-, vascular endothelial-, pancreatic- and neurallike cells (Strom and Miki, 2003). Should this finding be confirmed, these ‘‘placental derived stem cells’’ (PDSC) offer a promising source of stem cells for both therapeutic and toxicological applications. However, there is currently no published data reporting the actual number of PDSC that can be derived from a single placenta. Further, due to the highly unusual properties of invasive placental cytotrophoblasts, the long-term safety of PDSCs needs to be carefully evaluated (Table 3). Derivation of hepatocyte-like cells from human placenta Because of the high level expression of drug metabolising enzymes, human hepatocytes are useful for the investigation of drug metabolism, toxicology and for studies of molecular genetics of drug metabolism pathways. In clinical trials, isolated and transplanted human hepatocytes have been used as a physiological and biochemical support of Table 3 Advantages and open questions of placental stem cell use Advantages

Open questions

Unlimited source

Tumour-like properties of placental cytotrophoblasts The actual number obtained from a single placenta may be low Proliferation capacity in long term culture not established, theoretically good Full differentiation capability not established, are multipotent and can differentiate into several specialised cell types

No ethical concerns No religious considerations

No legislative regulations

Non-invasive for donors No risks for donors


liver function or as a cell therapy for metabolic defects in liver function (Millis et al., 2002; Patzer et al., 2002). PDSCs seem to be able to differentiate along a hepatic lineage as determined by the expression of the epithelial cytokeratins 8 and 18, and the expression of genes characteristic of hepatic differentiation such as albumin and alpha-1 anti-trypsin (A1AT). Hepatocyte-like cells derived from placental stem cells express a number of the CYP450 genes suggesting that the cells differentiate fully along the hepatic lineage. The presence of mature hepatic gene expression in placental-derived hepatocytes suggests that these cells could be useful in applications where adult human hepatocytes are currently utilised, including toxicology and drug metabolism and even in the cell therapy of liver disease (Strom and Miki, 2003). It has been reported to be feasible to isolate PDSCs also from the amniotic membranes, although placenta seems to be a more abundant source (Strom and Miki, 2003). Other groups have reported isolation of mesenchymal cells from amniotic membranes or amniotic fluid and their differentiation into chondrocytes, neural cells and insulinproducing cells (Bossolaco, 2004; Nikaido, 2004; Wei et al., 2003) (Table 1). Currently, PDSCs have not been tested in published preclinical or clinical investigations. On the other hand, there are several reports describing the use of amniotic epithelial cells in tissue repair. Since these cells do not express HLAmolecules on their surface, they are non-immunogeneic and have been recognised as a potentially promising source of allogeneic graft material. The use of amniotic membrane in the treatment of human corneal injuries (e.g., chemical burns) has resulted in good clinical outcome and it is practised by several centers (Shimmura and Tsubota, 2002). In this application, the amniotic membrane is thought to secrete growth factors promoting the regeneration of the patients’ own cornea from his/her limbal stem cells rather than leading to corneal differentiation of the amniotic cells. However, amniotic epithelial cells have also been reported to display neural characteristics including expression of neural genes (e.g., nestin), secretion of neurotrophins (e.g., BDNF) and synthesis of neurotransmitters (dopamine and acetylcholine) (Kakishita et al., 2000, 2003; Okawa et al., 2001; Sakuragawa et al., 1997). Human amniotic epithelial cells have been tested in a monkey model of spinal cord injury and demonstrated to integrate into site of injury and allow the penetration of the cell graft by host axons (Sankar and Muthusamy, 2003). In a rat model of Parkinson’s disease, they have been reported to survive in rat brain, produce dopamine and provide partial amelioration of the disease (Kakishita et al., 2003). Further, in a rat model of brain ischemia, rat amniotic epithelial cells were shown to transform into neuron-like cells in the ischemic area (Sakuragawa et al., 1997). Amniotic epithelial cells may thus have potential as a cell source for cellular therapies especially aimed at neural diseases or injury (Table 2).


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Use and application of stem cells in toxicology Mutagenic, embryotoxic and teratogenic substances may exert direct cytotoxic effects and induce alterations of embryonic development as a result of mutations at the DNA level. Additionally, developmental defects may be generated by interference of mutagenic or embryotoxic substances with regulatory processes of proliferation and differentiation. Numerous well-established in vivo test systems using laboratory animals have been utilised for mutagenicity testing and reproductive toxicology. These experiments require relatively large number of animals, are expensive, laborious and time-consuming, since the studies many times include pre-conceptional, pre- and postnatal phases and multigenerational follow-up. On the other hand, in vivo experiments best mimic the normal physiology of the organism. To reduce the number of animal experiments, in vitro alternatives such as primary cell and organ cultures and permanent cell lines as well as whole embryo cultures, have been developed (Davila et al., 1998; Piersma, 2004). While cell, organ and embryo cultures are well established as cellular screening models in toxicology, they exhibit several limitations. In many cases, the primary cultures or established cell lines do not represent the functional properties of specialised somatic cells. In vitro culture often results in a loss of proliferation capacity, viability and tissue-specific properties during long-term cultivation. Further, cellular systems are not able to recapitulate developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach to generate functional cell types in vitro is offered by stem cells (Davila et al., 2004; Rohwedel et al., 2001; Rolletschek et al., 2004). Stem cell-technology provides unprecedented opportunities for identifying new molecular targets, discover and develop new drugs, and for testing them for safety. Due to the self-renewing property of stem cells, they can theoretically be continuously cultured in an undifferentiated state and give rise to more specialised cells of the human body. Many recent studies have indicated that during in vitro differentiation, stem cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis and result in functionally competent specialised cell types. Although morphogenetic development is not possible in stem cell models due to lack of spatially controlled signals, cellular differentiation occurs in a developmentally regulated and time-dependent manner. Thus, stem cell models are suitable for analyses of developmental processes on a cellular level, which is a prerequisite to determine effects of embryotoxic and teratogenic factors in vitro. During the last years, stem cells have been used in several applications including pharmacological, cytotoxicological and embryotoxicological approaches. However, at present, applications using stem cells in basic and applied toxicology are still preliminary. The combination of stem

cell models with the latest innovations in genomics and proteomics techniques for the identification of target genes and molecules influenced by application of test compounds, as well as application of new strategies for genetic and epigenetic manipulation of cells is likely to open new perspectives in basic and applied toxicology (Davila et al., 2004; Rohwedel et al., 2001; Rolletschek et al., 2004). At the moment, the best alternative for conducting mechanistic studies and risk assessments of putative biohazards and new drugs for human toxicity in vivo is still to use immunodeficient mice housing grafted human tissues. This model accounts for both the route of exposure and metabolism of the compound, and has been successfully used to study the effects of tobacco smoke-derived aromatic hydrocarbons in human oocytes (Matikainen et al., 2001).

Acknowledgments This work was supported by grants from the Research Funds from the University Central Hospital in Helsinki, The Finnish Medical Foundation and Foundation for Diabetes Research (T.M.) and from the Academy of Finland (J.L.).

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