ArticleDerivation efficiency, cell proliferation, freeze–thaw survival, stem-cell properties and differentiation of human Wharton’s jelly stem cells
Introduction
Human stem cell biology has drawn tremendous interest recently. Stem cells have been isolated from preimplantation embryos, fetuses, umbilical cords and adult organs (Bongso and Lee 2005) and, more recently, by reprogramming somatic cells to the embryonic state (human induced pluripotent stem cells; iPSC) (Takahashi et al., 2007, Wernig et al., 2007). Based on the presence of specific markers they have been classified as embryonic stem cells (ESC), haematopoietic stem cells (HSC) and mesenchymal stem cells (MSC). The plasticity of these stem cell types appears to be varied. Human ESC and iPSC are pluripotent in that they are able to differentiate into almost all the tissues of the human body (Luong et al., 2008) while human MSC and HSC are multipotent being able to differentiate into a few lineages (Zapata, 2009).
Although human ESC and iPSC are pluripotent, their clinical application has thus far been hindered by the problems of inadequate cell numbers, possible teratoma formation or immunorejection after transplantation (Carpenter et al., 2009, Luong et al., 2008). Various research studies are being attempted to overcome these hurdles (Bongso et al., 2008). Human MSC, though not as pluripotent as human ESC, are, however, non-controversial and have been isolated from adult bone marrow, fetal and adult organs, umbilical cord blood and from the umbilical cord matrix or stroma. Adult bone marrow is the most popular source of MSC for stem cell research and application, but their harvest is invasive and painful with the risk of infection and donor site morbidity, their numbers are small and decline with the age of the patient, their stem-cell properties are retained only for a few passages in vitro, they possess poor expansion capacity and their use is currently limited to only autologous treatment (Mueller and Glowacki, 2001, Troyer and Weiss, 2008).
The existence of MSC in human umbilical cord blood (UCB) has been controversial. Mareschi et al. (2001) reported that human MSC could be isolated from bone marrow but not from UCB and Wexler et al. (2003) claimed that adult bone marrow was a rich source of MSC whereas UCB and peripheral adult blood were not. However, other investigations have shown the presence of human MSC in UCB (Lee et al., 2004; Secco et al., 2008). Musina et al. (2007) reported that the typical features of human MSC in UCB were their low counts per volume of UCB and very low proliferation rates. UCB-derived human MSC have been differentiated into chondrocytes (Wang et al., 2004), neuron-like cells (Hou et al., 2003) and endodermal tissues such as liver (Anzalone et al., 2009). However, inadequate cell numbers and poor growth rates are a major impediment in the use of human MSC from UCB for regenerative medicine purposes for the patient (Musina et al., 2007).
The human umbilical cord originates from the extraembryonic mesoderm, which in turn is derived from the proliferation of the epiblast and has within it two arteries and a vein. The matrix, which is the compartment between the umbilical blood vessels and amniotic wall of the cord, is a rich source of MSC. MSC have been derived from various parts of this matrix using different derivation protocols and these have been given various names. MSC populations were reported from the perivascular regions around the blood vessels (perivascular stem cells; Sarugaser et al., 2005), from the inner amnion membrane (subamnion stem cells; Ruetze et al., 2008), from the outer amnion membrane (human amnion membrane stem cells; Illancheran et al., 2007), the intervascular compartment (human umbilical cord matrix stem cells; Weiss et al., 2006) and from the Wharton’s jelly (human Wharton’s jelly stem cells; WJSC; Fong et al., 2007). The cells from all these compartments were shown to possess stem-cell properties. However, their growth behaviour, characterization and differentiation potential have not been compared between each other and with other human MSC types so as to evaluate their true therapeutic potential. Human umbilical cords can range from 50 to 60 cm in length and are discarded at birth and, as such, MSC derived from them are non-controversial and can be harvested in large numbers for research and therapeutic use. Human WJSC possess both mesenchymal and embryonic stem cell markers, have prolonged self-renewal, are non-tumorigenic and are tolerated in allogeneic transplantation (Troyer and Weiss, 2008, Fong et al., 2009, La Rocca, 2009, Mitchell et al., 2003). They could be differentiated into pancreatic islets, neurons, cartilage and bone (Chao et al., 2008, Fu et al., 2006, Hou et al., 2009, Wang et al., 2009, Weiss et al., 2006) and when these human WJSC-derived tissues were transplanted into diseased animal models, they engrafted and showed improved functional outcome (Chao et al., 2008; Weiss et al., 2006).
Since the gelatinous Wharton’s jelly can be separated with minimal contamination from the rest of the constituents of the umbilical cord, this detailed study was undertaken with respect to the human MSC within it and the cell recovery and expansion rates, retention of stem-cell properties in prolonged culture, freeze–thaw survival and differentiation potential with the view of evaluating their benefits over human UCB MSC for concurrent banking with cord blood for future regenerative medicine purposes.
Section snippets
Derivation, growth and population doubling times of human WJSC
Institutional Domain Specific Review Board approval with informed patient consent was given for the derivation of WJSC from human umbilical cords of single and twin pregnancies. Approximately, 7.5 cm length pieces cut from the mid-region of each of 13 human umbilical cords were collected in sterile transport medium (Hank’s balanced salt solution; Invitrogen Life Technologies, Carlsbad, CA, USA), coded for anonymity and stored at 4°C for 2–6 h before processing. Each piece of umbilical cord was
Morphological characteristics, growth and population doubling time of human WJSC
Human WJSC were successfully derived and propagated to produce cell lines from all 13 umbilical cords (100%) using the derivation method described in this study. The length of each piece of umbilical cord collected was 7.5 cm and approximately 3.5 ± 0.6 × 107 fresh live WJSC were obtained from each piece (4.7 ± 0.2 × 106 cells/cm). The cells were divided equally into simple and complex media (1.75 ± 0.4 × 107 cells per medium) and used to seed three or four 100 mm Petri dishes per medium.
Discussion
Human Wharton’s jelly stem cells are adherent cells that attach very well to plastic surfaces both in primary culture and passages and thus do not require feeders for their derivation and propagation, unlike human ESC. The mean number of adherent human WJSC in culture in the present study was much higher than that reported by Lu et al. (2006) perhaps due to the richer complex medium used. Since the PDT was slower in simple medium, it is important that complex media be used when human WJSC are
Acknowledgements
The authors acknowledge the grant support provided by the National Medical Research Council of Singapore (R-174–000–103–213) and the National University of Singapore (R-174–000–089–133).
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Dr Chui-Yee Fong is an Assistant Professor in the Department of Obstetrics and Gynaecology of the National University of Singapore. With a background in IVF, she has over a decade of research experience with stem cells of the reproductive system. She was involved in the first isolation of human embryonic stem cells (ESC) in 1994, later the derivation of research- and clinical-grade ESC lines and recently the derivation of human umbilical cord Wharton’s jelly stem cell (WJSC) lines. Her current interests include the differentiation of WJSCs into desirable tissues and the evaluation of their anticancer effects.