1 Introduction

The engineering of stem cells to restore defect functions in human tissues is an exciting challenge in the field of regenerative medicine. Embryonic stem cells (ESC) have a great potential for this purpose due to their pluripotent differentiation capability, but their use is limited by serious ethical considerations. Recent findings evidencing the reprogramming of somatic cells to pluripotent stem cells (termed IPS cells) [1] open ethically acceptable perspectives. However the concern remains that undifferentiated IPS cells as well as ESC may form teratomas after transplantation in the body.

Adult mesenchymal stem or stromal cells (MSCs) are considered a valuable alternative to these cells. Since their discovery in bone marrow (BM) by Friedenstein et al. [2], BMSCs have been extensively investigated and their use in animal studies as well as in clinical trials showed encouraging results (reviewed in [3]). Today BM-MSCs are still considered as the “gold standard” for the use of adult MSCs. Nevertheless BM as a source for MSCs presents several disadvantages. Besides the invasive and painful collecting procedure, in BM-aspirates MSCs are present at very low frequency (approximately 0.001–0.01% [4]) and their quality varies with the age of the donor. The low frequency implies that an extensive in vitro expansion of the cells will be required to deliver clinical doses to a patient, which enhances the risk of epigenetic damages as well as viral and bacterial contaminations. For these reasons, alternative sources of MSCs are needed.

In this context, the human umbilical cord (UC) gained more and more attention during the last decade (see Fig. 1). The UC is a non-controversial and accessible source of autologous cells, which can be easily processed after birth. It has been demonstrated that MSCs are found both in the blood (UCB) [5] and in the tissues of UC. However, UCB-derived MSCs may have a limited technological potential because their frequency seems even lower than in BM (range 0.001–0.000001% [6]) and their isolation is hardly reproducible [7, 8], whereby UCB contains larger amounts of other tissue stem cell populations including CD133+ cells or hematopoietic stem cells (CD34+). In contrast, the frequency of MSCs in UC-tissues is believed to be much higher. Thus, using robust isolation procedures, a large number of multipotent primitive stromal cells with high proliferation capacity can be isolated. All these features open interesting perspectives for the scalable production and engineering of UC-derived cells for clinical applications. Here, we give an overview of the scientific evidences collected during the recent years that human UC may be a valuable cell source for cell-based therapies.

Fig. 1
figure 1

Cumulative number of publications over the last 15 years dealing with UC-derived MSCs (entries by PubMed with the terms “mesenchymal stem cells” and “umbilical cord” till July 2009)

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2 The Human Umbilical Cord: A Source of MSCs

The human UC is the lifeline between the fetus and the placenta. It is formed during the fifth week of embryogenesis and grows to a final length of approximately 60–65 cm, weighs about 40 g, and has a mean diameter of 1.5 cm in normal pregnancies [9, 10]. UC usually comprises two arteries and a vein, which are immersed within the so-called Wharton’s jelly (WJ) and enclosed by a simple amniotic epithelium (see Fig. 2a). WJ is a mucoid connective tissue rich in proteoglycans and hyaluronic acid (HA), which insulates and protects umbilical vessels from torsion, compression, or bending and therefore ensures a constant blood flow between fetus and placenta.

Fig. 2
figure 2

Cross section of an umbilical cord. (a) UC consists of two arteries and one vein embedded in the Wharton’s jelly and surrounded by an amniotic epithelium (modified from [95]). (b) Four separate compartments within the umbilical cord have been shown to comprise mesenchymal stromal cells

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In recent years, several studies described at least four separate regions (see Fig. 2b) of the UC containing MSCs. The term “MSC” has been related to several definitions. In this chapter we use “MSC” as an acronym for mesenchymal stromal cell (discussed in Sect. 4). MSCs could successfully be isolated from UCB [7, 11–15], the umbilical vein subendothelium [16–18], the intervascular region [19–29], the perivascular region [30, 31], or from whole UC tissue [32, 33]. UC-derived MSCs meet the basic definition of multipotent MSCs as postulated by the International Society for Cellular Therapy (ISCT) (see Sect. 4).

Thus, this chapter will focus on MSCs derived from UC tissue, but not from UCB (see Fig. 2b, region 2–4).

3 Isolation of MSCs from the Umbilical Cord

In recent years, several investigators published protocols for isolating MSCs from the UC tissue. Depending on from which part of the cord the cells should be isolated, protocols have been adopted and modified. A schematic overview of applied isolation protocols is given in Fig. 3. Basically, the isolation procedure starts with the removal of umbilical vessels. The cord is then cut down to smaller segments or chopped into small pieces which are subsequently enzymatically digested [22, 23, 29]. Alternative isolation methods without removal of vessels [34, 35] and without enzymatic digestions [26, 34] or explant cultures [33, 36] have also been described. To isolate cells from the perivascular tissue or the subendothelium of the umbilical vein, further methods have been established [16–18, 30, 31].

Fig. 3
figure 3

Schematic overview of applied isolation protocols. Various approaches have been used to isolate mesenchymal stromal cells from the umbilical cord tissue. Basically, the isolation procedure includes steps of removing umbilical vessels, tissue chopping and enzymatic digestion (indicated by bold arrows), but several alterations of protocols have been described

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We have used a protocol without enzymatic digestion and without removal of umbilical vessels to isolate MSC-like cells from whole UC tissue in an explant culture approach. Therefore, human UCs were obtained from patients with written consent delivering full-term (38–40 weeks) infants by cesarean section. The use of this material has been approved by the Institutional Review Board, project #3037 in an extended permission on June 17, 2006.

First, blood from arteries and the vein was removed by flushing phosphate buffered saline (PBS) through the vessels using a sterile syringe and blunt needles. Thereafter, UC was stored in an appropriate transfer medium (PBS) enriched with 5 g L−1 glucose, 50 μg mL−1 gentamicin, 2.5 μg mL−1 amphotericin B, 100 U.mL−1 penicillin, and 100 μg mL−1 streptomycin), to minimize the risk of contaminations. The UC was first cut into 10–15 cm long segments which were subsequently cut into approximately 0.5 cm3 large pieces. During the isolation procedure, transfer medium was used to keep the cord and the minced pieces moist. Finally, the small pieces were transferred to cell culture flaks (Fig. 4a) and incubated in αMEM supplemented with 15% of allogous human serum and 50 μg m−1 gentamicin at 37°C in a humidified atmosphere with 5% CO2. The medium was changed every second day. An outgrowth of adherent cells from single tissue pieces could be observed after approximately 10 days (Fig. 4b). After 2 weeks the UC tissue was removed and the adherent cells (Fig. 4c) were harvested by enzymatic treatment. The obtained cell suspension was centrifuged at 200 g for 5 min and the cells were resuspended in αMEM supplemented with 10% human serum and 50 μg mL−1 gentamicin and subcultured at a density of 4000 cells cm−2. These culture conditions have demonstrated to support an optimal growth of the cells.

Fig. 4
figure 4

Isolation of mesenchymal stem cell-like cells from umbilical cord. (a) Explant culture of minced UC tissue. (b) After approximately 10 days of culture cells start to grow out of the small UC segments. (c) Adherent growing monolayer of fibroblast-shaped cells after 2 weeks of culture

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The isolated cells exhibited a high proliferation potential. Cell population doubling times ranged from 27.5 ± 1.6 h (passage 2) to 78.9 ± 6.3 h (passage 17). At our culture conditions, the cells could be expanded without loss of proliferative activity and viability at least for 20 population doublings. After approximately 50 population doublings the cells entered a phase of replicative senescence. High proliferation potential and expansion capacity are common features for UC-derived stromal cells, which were described by several other groups [20, 26, 29, 30, 34, 35, 37, 38]. Furthermore, UC-derived cells could be efficiently cryopreserved and revitalized. We used a cryo-medium containing 80% human serum, 10% culture medium, and 10 % DMSO. The cells were gradually frozen at a rate of 1°C min−1 and finally stored at −196°C. At these conditions cell survival rate after rapid thawing at 37°C reached 75 ± 12.8%.

To date, it still remains to be further investigated whether cells isolated from different compartments or derived by different isolation procedures share the same stem cell characteristics, e.g., proliferation and differentiation potential and immunologic properties (see below).

4 Characterization of UC-Derived MSCs

The acronym “MSC” has been widely used in the literature for “mesenchymal stromal cell” as well as for “mesenchymal stem cell” to denominate plastic-adherent fibroblast-like cultures isolated from different adult or extra-embryonic tissues. Because there is currently no consensus set of markers allowing the identification of MSCs and considering the fact the definition criteria for stem cell is not unanimously accepted [39], it appeared unwise to apply the term “stem cell” for mesenchymal cell population. In this context, ISCT proposed to term plastic-adherent fibroblast cultures “multipotent mesenchymal stromal cells” (MSC) [40] and published in 2006 the minimal criteria defining these cells [41].

UC-derived stromal cells meet the basic criteria defined by the ISCT, namely the adherence to plastic, the expression of a set of specific surface antigens (see below), and a multipotent differentiation potential (discussed in paragraph 5 Differentiation Potential).

Histologically, cells freshly isolated from the UC are mainly fibroblastic in appearance (see Fig. 5a). However, some groups reported more than one phenotype in UC-derived MSC cultures [23, 29, 31, 36] and noticed changes in the distribution of the phenotype after several passages [23, 36]. Our group observed for instance a broad cell size distribution and marked morphological differences in isolated UC-MSCs cultures (see Fig. 5b). After fractionation of different populations via counterflow centrifugal elutriation (CCE) according to the size of the cells, we obtained two sub-populations with significant differences in cell size, growth properties, and biochemical markers expression. Whereas small-sized subpopulation exhibited the highest proliferative capacity and the most pronounced expression of MSC markers, large-sized cells were identified as senescent via β-galactosidase staining (see Fig. 6) [36]. These findings may be of importance in order to deliver high quality cells for clinical applications.

Fig. 5
figure 5

Morphology of UC-derived stroma cells. (a) UC MSC show predominantly fibroblastic morphology. The cytoplasm of cells was visualized via Calcein-AM stain (100× magnification). (b) Cells with marked morphological differences can be rather observed in the cultures collected from UC. The image presents large cells (a, white arrows) surrounded by small cells (b, black arrows), which show increased nucleus-to-cytoplasm ratio. For the visualization of cell nuclei DAPI staining was performed (200× magnification)

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Fig. 6
figure 6

Senescence staining in sub-population of UC-derived primary cell cultures [36]. (a) UC-derived primary cell population. (b) CCE-derived subpopulation of small-sized cells. (c) CCE-derived subpopulation of large-sized cells. (d) Comparison of the senescence level in the CCE-derived fractions. Cells were cultured for 6 days after elutriation. Following subculture, the cells were seeded at a density of 6000 cells cm−2 and cultured for further 48 h in complete medium. A relative high portion of multi-nucleated cells (arrows) were detectable in the subpopulation of the large-sized cells. Student’s t-tests were performed for the recognition of the significant differences (marked with asterisks) in comparison to UC-derived primary cell population

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An additional feature of MSCs is their clonogenicity. A single cell is able to rise to a fibroblastic colony in a so-called colony forming unit fibroblast (CFU-F) assay. Historically, this characterization parameter is linked to the pioneer work of Friedenstein et al., who first isolated stromal cells from BM according to their capability to form fibroblastic colonies and demonstrated their osteogenic potential in vitro [2]. The CFU-F assay gives the frequency of fibroblast-like cells within a population liable to extensive proliferation and to rise to a colony (see Fig. 7).

Fig. 7
figure 7

CFU-F assay of UC-derived stromal cells

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This approach is commonly used to enumerate MSCs in a particular tissue [4]. For instance, Lu et al. recently evaluated the frequency at 1 CFU-F per 1609 mononuclear cells (MNCs) in whole UC tissues [35]. More specifically, 1 CFU-F per 333 MNCs was reported in cells isolated from perivascular tissues of the UC vein [31]. In comparison, the isolation frequency of CFU-F from BM is estimated in a range of 1–10 CFU-F per 105 MNCs [4] and only 1 CFU-F per 108 MNCs [7] to 1–3 CFU-F per 106 MNCs are reported in UCB [42, 43]. According to these data, the human UC is considered to harbor a higher number of MSCs than found in BM or UCB. The results of the CFU-F assay, however, depend on different parameter such as the isolation method, culture conditions, as well as the cell seeding density. This leads to a high degree of variability in the results and makes the comparison of the published data difficult. Analysis of specific molecule expression at the single cell level via flow cytometry is strongly advisable to identify MSCs within a mixed cell population.

In contrast to other progenitor cell populations, such as, for instance, hematopoeitic stem cells, there is currently no specific marker available defining human MSCs. The expression of a set of markers combined with the demonstration of in vitro multi-lineage differentiation potential is necessary to identify MSCs in UC-derived cell populations. Table 1 summarizes extracellular and intracellular molecules expressed by UC-MSCs reported in the literature up to July 2009.

Table 1 Reported intra- and extra-cellular markers of UC-derived MSCs till July 2009
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The surface antigen SH2 (CD105), SH3 (CD73), and Thy-1 (CD90) are widely used for the identification of UC-derived stromal cells (see Table 1), as these markers are proposed by the ISCT as positive markers for human MSCs [41]. However, these epitopes are also expressed on hematopoietic and endothelial cells, which are two potential contaminants in UC-derived cell populations. Consequently, it is necessary to carefully exclude cells from hematopoietic or endothelial origin using surface marker such as CD45, CD34, or CD31. HA receptor CD44 is also a commonly accepted marker, as the extracellular matrix of the UC is one of the highest HA-containing tissue in humans [44]. Figure 8 exemplarily illustrates the immunophenotype of a stromal cell population isolated from whole UC tissue by our group. Additionally, like MSCs isolated from other tissues, UC-derived stroma cells do not express the human leukocyte antigen HLA-DR but express HLA-I. However, Sarugaser et al. reported that the expression of the latter marker may be manipulated in vitro, which may be very promising in term of allogenic transplantations [31].

Fig. 8
figure 8

Flow cytometric analysis of UC-derived stroma cells

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UC-derived stroma cells were found positive for pluripotency markers usually expressed by ESCs such as Oct-3/4, Nanog, Sox-2, or SSEA-4 (see Table 1), which underlines their primitive nature. The primitive character of the UC-derived cells is also illustrated by their high proliferation and expansion capacity. UC-derived stroma cells have shorter doubling times compared to adult BM-MSCs [30, 35, 37, 38], exhibit telomerase activity [23, 26, 45], and could be expanded in vitro to a number of population doublings ranging from 20 to 80 without evidences of senescence or abnormal karyotype [20, 26, 29, 34]. It was first unclear whether UC-derived stroma cells were homogenous regarding their primitiveness or if UC-derived stroma populations rather harbor a subset of primitive MSCs [46]. For instance, population doubling times estimated between 60 and 85 h for freshly isolated UC-cells rapidly decrease within 2–3 passages to approximately 25 h [23, 31], which may indicate the presence of a fast growing sub-population of more primitive cells overgrowing the initial population. This hypothesis was further strengthened by recent works demonstrating via flow cytometry a subset of cells expressing pluripotency markers [47, 48]. Zhang et al., for instance, reported that approximately 20% of stroma cells isolated from perivascular tissues of the umbilical arteria express Oct-3/4 and Nanog [48].

With growing evidence that MSC-like cell population isolated from UC tissues are rather heterogeneous, at least in regard to primitive marker expression, the identification of a universal marker defining primitive human MSCs remains challenging. Several cell surface molecules were recently proposed for the identification and isolation of MSCs in BM aspirates such as CD271 [49, 50], MSCA-1 [50], SSEA-4 [51], and the neural ganglioside GD2 [52, 53]. To our knowledge, CD271 and MSCA-1 expressions have not been reported yet in UC-derived stroma cell populations. Xu et al. recently isolated a subset of GD2+ cells exhibiting a high clonogenicity as well as proliferation capacity but also a significantly stronger multi-differentiation potential than GD2− cells. According to these results, GD2 may be a useful marker to isolate multipotent MSCs from UC-tissues, but further studies are needed to verify these findings.

The most convincing biological property for the identification of MSCs remains the capability to differentiate into mesodermal lineages. In the next section the in vitro differentiation potential of UC-derived stromal cells will be discussed.

5 In Vitro Differentiation Potential

The differentiation repertoire of stroma cells derived from UC tissue reported in the literature till July 2009 is summarized in Table 2.

Table 2 Differentiation potential of stroma cells derived from human umbilical cord tissue reported in the literature till July 2009
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The potential of UC stroma cells to differentiate into adipocytes, chondrocytes, and osteocytes has been widely investigated and well established by several groups. According to the minimal definition criteria proposed by the ISCT, UC-derived stroma cells are considered multipotent MSCs [41]. Successful adipogenic, chondrogenic, and osteogenic differentiation of UC-derived MSCs are presented in Fig. 9.

Fig. 9
figure 9

Adipogenic, chondrogenic and osteogenic potential of UC-derived MSCs. (a) Formation of lipid droplets stained with oil red O in Wharton’s jelly cells after adipogenic induction, scale bar = 20 µm (modified from [23]), (b) cell sphere obtained in droplet culture of chondrogenically induced UC-MSCs (scale bar = 500 µm) with abundant type II collagen expression (in c, scale bar = 50 µm) (modified from [23]), (d) ALP expression after osteogenic differentiation of umbilical vein derived MSCs (modified from [17]). (e) Mineralization of osteogenically induced culture of umbilical vein derived MSCs evidenced by von Kossa staining (modified from [17])

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Adipogenic potential is usually demonstrated by the apparition of cells exhibiting intracellular lipid droplets (Fig. 9a). The capacity to form chondroblasts is evidenced by the formation of shiny cell-spheres with type II collagen expression in the extracellular matrix in droplet cultures (Fig. 9b). Enhanced ALP expression and mineralization assayed by von Kossa or alizarin red staining demonstrate osteogenic potency (Fig. 9d, e). It should also be mentioned that sub-populations of cells spontaneously exhibiting a functional osteogenic potential with mineralized bone nodules can be observed in UC-MSCs cultures [31]. Such bone nodules are presented exemplary in Fig. 10.

Fig. 10
figure 10

Mineralized bone nodule in UC-MSCs culture. (a) Phase contrast microscopy picture of a bone nodule, (b) alkaline phosphatase (violet dark cells) and alizarin red staining of a nodule (arrow), (c) alkaline phosphatase (violet dark cells) and von Kossa staining of a nodule (arrow)

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In addition, it has been shown that UC-MSCs can successfully differentiate to endothelial cells after addition of VEGF and b-FGF [54, 55] and can form vessel-like structures in matrigel cultures [37, 55]. Furthermore, some UC-derived cell populations also seem to be able to differentiate to muscle cells. For instance, WJ cells (WJCs) could be induced to skeletal myocytes when placed in myogenic medium [20]. Differentiation to cardiomyocytes was also reported but remains controversial. Whang et al. demonstrated for instance that WJCs could be induced to cells exhibiting cardiomyocyte morphology and expressing specific markers (N-cadherin and cardiac troponin) using 5-azacytidine or cardiomyocyte-conditioned medium [28]. Kadivar et al. observed cardiomyocyte like cells expressing cardiac specific genes after 5-azacytidine induction of UC-MSCs isolated from the endothelium/subendothelium layer of the UC vein. In contrast to these results, Martin-Rendon et al. could not detect cardiac markers expression after in vitro induction of MSCs isolated from the WJs and perivascular tissues [56]. Furthermore, differentiated in vitro cultures of functional cardiomyocytes presenting beating clusters are poorly or not demonstrated. To our knowledge, only one group reported differentiated cells exhibiting slight spontaneous beating after 21 days of induction; however no quantitative data are presented in this study [57].

Recent findings suggest that UC-MSCs can differentiate into endodermal lineages. Campard et al. reported that UC-matrix cells constitutively expressed markers of hepatic lineage, such as albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. After in vitro hepatic induction, cells exhibiting a hepatocyte-like morphology with hepatic features such as specific markers up-regulation and urea production were observed. However, the authors pointed out that their cells lack important characteristics of functional liver cells and thus conclude that UC-matrix cells can be differentiated at least to immature hepatocytes [58]. Chao et al. were also able to induce WJCs using a four stage differentiation protocol to form islet-like clusters expressing pancreatic related genes and secreting insulin in response to glucose concentrations [59]. Recent results from Wu et al., who successfully differentiated WJCs to pancreatic cells and observed higher differentiation potential compared to BM-MSCs [38], further reinforce these findings.

Finally, several groups observed the differentiation of WJCs to cells exhibiting morphological and biochemical characteristics of neural cells, suggesting that UC-MSCs are able to differentiate to a certain state of maturation along the neuronal lineage [21–23, 25, 26, 35, 60–62]. Mitchell et al. were the first to observe neuronal differentiation of WJCs after stimulation with b-FGF and other neuronal differentiation reagents [26]. The differentiation was attested according to morphological changes and expression of neuron-specific enolase, βIII-tubulin, neurofilament M and tyrosin hydroxylase [26]. The differentiation potential was then confirmed by several other groups [21, 23, 25, 60]. Figure 11 shows exemplary neuronal cells obtained by Karahuseyinoglu et al. after neuronal induction of a sub-population of WJCs [23]. Interestingly, it also seems possible to generate some sub-types of neurones as demonstrated by Fu et al., who were able to obtain dopaminergic neurones from WJCs [21].

Fig. 11
figure 11

Neuronal differentiation of WJCs, modified from [23]. (a) β-III Tubulin expression, (b) Nestin expression located in the perinuclear cytoplasm in particular (b′), (c) neurofilament-160 (NF-M), (d) neuron-specific nuclear protein expression (Neu-N) restricted to the nucleus, (e) neuron-specific enolase (NSE), (f) microtubule-associated protein-2 (MAP2) detected as discontinuities along the cells. (g) MAP2 distribution in cell–cell contact. Scale bars = 10 µm (b′), 20 µm (b, c), 50 µm (e), 100 µm (a, c, d)

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Summarizing the published data, we find strong evidence to suggest that the human UC is a source of multipotent stroma cells which are capable of differentiating into mesodermal and non-mesodermal lineages. It remains unclear whether the differentiation potential of the UC-derived MSCs depends on their location in the UC-tissues. For instance, Suzdal'tseva et al. reported that only a few cells isolated from the cord vein subendothelial tissue were able to differentiate to osteoblasts [63]. In contrast, cells isolated from perivascular tissues of the umbilical vein showed a high osteogenic potential with spontaneous formation of bone nodules [31], which was even evaluated higher than the potential of bone-marrow MSCs in a comparative study [30]. Recently, two sub-populations were evidenced in cultures of WJ-derived MSCs with regard to the expression of vimentin and pan-cytokeratin filaments [23]. Interestingly, cells expressing cytokeratin, predominantly located in the perivascular tissue of the cord, did not differentiate into neurones in vitro. These findings are consistent with the results of Sarugaser et al., who showed that perivascular UC-cells could not be induced to the neuronal lineage [31]. The hypothesis of a location-dependent differentiation potential of UC-derived stroma cells is also supported by the fact that a gradient of cell maturity was observed within the UC tissues [64]. According to the cytoskeletal complexity, the most immature cells are located in subamniotic and intervascular regions, whereas cells of perivascular regions may represent a more differentiated state [64, 65].

Many groups most likely investigate mixed populations of UC-MSCs, particularly if the cells are derived from whole UC or from the WJ. Thus, the results of studies comparing the differentiation potential of UC-derived MSCs with other sources (for example BM) should be carefully interpreted [17, 56, 66, 67]. More work is needed to attest whether cells isolated from a defined compartment of the UC is more suitable for a specific differentiation lineage. This information would be of tremendous importance for clinical applications of UC-derived MSCs.

6 Immune Properties of MSCs and In Vivo Applications

Besides their multi-lineage differentiation potential, BM-derived MSCs have been shown to exhibit immune-privileged and immune-modulatory properties, which predestine them as ideal candidates for cell-based therapies. They fail to induce proliferation of allogeneic lymphocytes in vitro and do not induce an immune response when used in allogenic mismatched animal experimental models [68–70]. Furthermore, they have regulatory effects on several cells of the immune system (e.g., T, B, dendritic, and natural killer cells) [71–77], prolong skin graft survival [78], and have been used in clinical applications to reduce acute and chronic graft-versus-host disease (GvHD) [79, 80]. Currently, three groups have investigated the in vitro immune properties of UC-derived MSCs and observed similar immunologic phenotypes to that of BM-MSCs. Ennis et al. [81] used cells isolated from the perivascular tissue of the UC [human UC perivascular cells (HUCPVC)] in one- and two-way mixed lymphocyte cultures (MLC) with resting or activated peripheral blood lymphocytes (PBL) to examine whether HUCPVCs induce or modulate proliferation of immune cells. Proliferation of PBLs was determined by measurement of 5-bromo-2-deoxyuridine (BrdU) or tritiated thymidine [3H] incorporation. They could show that HUCPVCs did not induce allogenic lymphocyte proliferation but reduced the proliferation of alloreactive PBLs in a dose-dependent way. Weiss et al. [82] describe similar observations using WJ-derived cells termed UC matrix stroma (UCMS) cells. In co-culture experiments they could show that UCMS cells not only suppressed the proliferation of Con-A-stimulated rat splenocytes [measured by live cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay and carboxyfluorescein diacetate succinimidyl ester (CFSE)-assay] and activated human peripheral blood mononuclear cells (PBMCs) or purified T cells (measured by tritiated thymidine [3H] incorporation) but also did not induce any proliferation of resting immune cells. Furthermore, flow cytometric analysis revealed the absence of the immune response-related surface antigens CD40, CD80, and CD86. Yoo et al. [83] compared the immune-suppressive effect of BM-MSCs and WJ-derived MSCs on phytohemagglutinin-induced T cell proliferation and report that both BM-MSCs and WJ-MSCs effectively reduced the proliferation of immune cells.

In vivo applications of UC-MSCs revealed further interesting attributes similar to BM-MSCs. Regarding their potential for cell-based therapy applications, UC-derived mesenchymal cells seem to support tissue repair by stimulating and modulating tissue-specific cells rather than differentiating into specialized cells. Yang et al. [84] reported a positive modulation of microglia and reactive astrocytes activities by UC-MSCs when transplanted into rats after complete transection of the spinal cord. They detected an elevated production of various cytokines around the lesion promoting spinal cord repair. Similar to these findings, Weiss et al. [29] hypothesized a supportive function of UC-MSCs mediated by various secreted trophic factors when used in a rodent model of Parkinson’s disease. Referring to their preliminary work on porcine UC-derived MSCs, which were successfully transplanted into rat brains without triggering an immune response or being rejected [85], they then transplanted human UC-MSCs into brains of Parkinson’s disease model rats without any immune-suppression. The transplanted cells did not produce brain tumors or a frank host immune rejection response. Furthermore, they significantly mitigated induced motor deficits [29]. Liao et al. [86] used UC-derived MSCs in a rodent stroke model and observed that the cells, injected into the rat brain, survived for at least 5 weeks and reduced injury volume and neurologic functional deficits of rats after stroke. They assume angiogenesis-promoting properties of the cells by producing angiogenic cytokines. Koh et al. [87] also applied a rodent stroke model. They used MSCs isolated from the umbilical vein sub-endothelium and induced differentiation of the cells into neuron-like cells, as indicated by morphology, expression of neuronal cell markers, and secretion of neurotrophic factors, before transplantation into rats. Since the UC-MSCs were both morphologically differentiated into neuronal cells and able to produce neurotrophic factors, but had not become functionally active neuronal cells, the authors hypothesize that the observed improvement in neurobehavioral function might be related to the neuroprotective effects of UC-MSCs rather than to the formation of a new network between host neurons and the implanted cells. Analogical findings were reported by Lund et al. [24]. They suggested a supportive behavior of MSCs in a rodent model of retinal disease when UC-MSCs were shown to contribute to photoreceptor rescue. The cells did not transform into neurons but more likely secreted neurotrophic factors, as indicated by higher expression levels of these factors in vitro.

Besides their supporting properties, UC-MSCs were also shown to be easily genetically manipulated. Friedman et al. [88] and Kermani et al. [89] both transfected UC-tissue derived MSCs with a GFP-reporter gene and created a stable cell line. Considering the immune-privileged and immune-modulatory properties, the cytokine production and supportive functions in vivo and the ability to be easily transfected, UC-derived mesenchymal cells are promising candidates for cell-based therapies and clinical applications. Currently, there are first clinical trials aiming to demonstrate if human UC-MSC have in vivo immune-suppressive effects and can be used for GVHD treatment [“Allogeneic Mesenchymal Stem Cell for Graft-Versus-Host Disease Treatment (MSCGVHD)”; ClinicalTrials.gov Identifier: NCT00749164; www.ClinicalTrials.gov].

7 Future Perspectives

In terms of cell engineering, the human UC is a very advantageous source of MSCs. Cells from UC are easily accessible, may be processed under GMP conditions, and the isolation of a high number of MSCs can be rapidly achieved in a reproducible manner. Particularly interesting features of UC-MSCs were evidenced in recent years. Due to their youth, UC-derived MSCs exhibit a high proliferation capacity and expansion potential. Thus, compared to other MSC-sources, for UC-derived MSCs no extensive expansion is required to obtain clinical doses, thereby reducing the risk of possible epigenetic damages occurring during the in vitro expansion process. Because one of the challenges of the bioprocesses will be the generation of clinical grade MSCs in disposable reactors, the monitoring of the cultures will be essential to control cell quality. The development of adequate in situ sensors for the monitoring of the cultures will be of great interest [90]. Furthermore, it has been shown that UC-MSCs can be frozen and thawed efficiently, which makes them suitable for their use in clinical cell banking. The therapeutic use of MSCs will require storage prior to clinical applications. In this regard, it appears worthwhile that UC-cells isolated at birth, may be safely stored and delivered decades later to a patient. Nevertheless, additional studies may be necessary to attest the stability of long term cryopreserved cells.

The clinical potential of MSCs is primary dependent on their differentiation potential. Like BM stromal cells, UC-derived MSCs were demonstrated to be multipotent. Interestingly, their differentiation repertoire does not seem to be restricted to the mesodermal lineages, since the cells could be successfully induced to neurones, liver, and pancreatic cells. A growing body of evidences suggests, however, that UC-MSC populations are rather heterogeneous, harboring a subset of primitive cells. The next generation of studies should focus on the identification and characterization of these sub-populations. In particular, the question of whether the differentiation potential of the isolated populations is dependent on their location in the UC-tissues is of great interest for clinical application. Newly described MSCs markers may be helpful in this regard.

Additionally, first in vitro and in vivo animal studies evidenced immune-privileged and immune-modulatory properties of UC-derived MSCs. Low levels of rejection were observed in all reports of in vivo transplantation experiments and encouraging results in tissue repairs were observed. In particular, supportive function through paracrine effects seems to be involved. The next generation of studies and first clinical trials will clarify whether the benefit of UC-derived MSCs after transplantation experiments relies on supportive effects and/or on differentiation in vivo.

One of the ambitious aims of regenerative medicine is the engineering of tissue in vitro. Few but very promising applications of UC-derived MSCs have been reported in this field. For instance, UC-MSCs are believed to have a high potential in cardiovascular tissue engineering [91]. They grew very well on bio-degradable polymer for the elaboration of cardiovascular constructs [33] and could be used for the construction of human pulmonary conduits [92], for the engineering of biologically active living heart valve leaflets [27], and for the elaboration of living patches with potential for pediatric cardiovascular tissue engineering [93]. The use of newly developed scaffolds, mechanical strain approaches, or 3D bioreactors for tissue generation, which were successfully applied with MSCs from other sources [94], will also be a highly interesting issue.

Considering the very encouraging results obtained in recent years, it may only be a question of time until UC-derived MSCs will be routinely used for clinical and tissue engineering applications.