How far are we from the clinical use of placental-derived mesenchymal stem cells?

Abstract

In recent years, multiple studies have investigated the biology and clinical applications of mesenchymal stem cells (MSCs), trying to define their markers, and elucidate their effects in animal models. MSCs are available from different tissues, and the use of placental-derived MSCs (PMSCs) for treating a variety of disorders is on the forefront. Herein, we discuss the most recent findings regarding the standardization of their isolation procedure and phenotype, along with advantages and limitations of their use. We also discuss the safety of the placental cell products, including the issue of senescence and mutagenesis of PMSCs, and efficacy from preclinical studies.

Keywords:

• isolation procedure
• phenotype
• placenta
• placental mesenchymal stem cells
• safety
• stability
• therapeutic application
• tumorigenesis

1. Mesenchymal stem cells in regenerative medicine

Mesenchymal stem cells (MSCs) were identified in 1970 in the bone marrow (BM) as fibroblast-like cells secreting factors involved in hematopoiesis [1] and with adipogenic, chondrogenic and osteogenic differentiation potential. Notably, MSCs can also be isolated from different placental tissues, including fetal membranes [2], different regions of the umbilical cord [3] and amniotic fluid [4].

There have been significant attempts to define markers, biological properties and therapeutic applications of MSCs [5]. MSCs are nowadays the most widely explored cell type for therapy due to their renowned regenerative and immune-regulatory properties [5]. The current need is to standardize in vitro assays in order to predict the potential therapeutic effects of different MSCs from different sources and to optimize protocols for clinical applications.

2. The issue regarding MSC sources: different sources, different uses?

MSCs are considered immune-privileged for their low expression of class II major histocompatibility complex and co-stimulatory molecules [2]. BM-MSCs possess immunomodulatory capacities [5], even though previous reports have shown that they can induce an immune response [6]. MSCs are available from different sources, thus possibly expanding their potential applications. However, this raises the question regarding which is the ideal source, considering also differentiation and functional properties of MSCs [7,8]. A major issue is the need to standardize a single isolation procedure for large-scale in vitro expansion, taking also into consideration whether it is possible to obtain enough cells from a single donor, or if it is necessary to use cells from multiple donors. Another concern is the choice between using allogeneic or autologous MSCs in clinical trials. The latter would allow better standardization and reduce production costs, since autologous cells are not always exploitable for efficient ex vivo expansion. As pointed out by Zhu and colleagues [9], this procedure has the intrinsic risk of genetic and epigenetic mutations that may lead to malignant transformation of MSCs derived from several tissues of different animal species. Deposition of epigenetic mutations was also discovered within placental-derived MSCs (PMSCs); however, these alterations did not translate into a malignant phenotype. This relevant issue has to be unraveled before any clinical application is pursued.

3. Placental mesenchymal stem/stromal cells

Human term placenta is an organ composed of different tissues. Cells with mesenchymal stem/stromal characteristics can be isolated from different regions of the fetal part of human term placenta, namely the amniotic membrane (human amniotic membrane-derived MSCs) and chorionic mesenchymal region (human chorionic mesenchymal stroma-derived MSCs), and also from the chorionic villi and regions of the umbilical cord [10]. Herein, PMSCs will be used as a general term and is referred to MSCs isolated from the placenta, not taking into consideration the specific region from which they were isolated.

3.1 Advantages and limitations

The advantages of placenta as an MSC source include its easy procurement with no harm to the mother and baby, high cell recovery, ethical acceptance, and interestingly, the possible higher stemness potential related to the early embryological origin of the placenta [11] and the unique immunomodulatory characteristics of placental cells [2]. Similar to MSCs from other sources, those derived from the human amniotic membrane inhibit T-cell proliferation in vitro [12] and in vivo [13], and MSCs from different placental regions have also been shown to strongly inhibit the generation, maturation and function of monocyte-derived dendritic cells [14]. Interestingly, the conditioned medium (CM) obtained from the culture of MSCs from the amniotic membrane possesses the same in vitro [8] and in vivo potential [15]. To the contrary, BM-MSCs and their CM exert immunomodulatory potential only if the cells are cultured in the presence of activating conditions [8,16]. This raises an interesting and advantageous point regarding the peculiar immunomodulatory characteristics unique to PMSCs, which constitutes an underlying mechanism of their therapeutic potential based on paracrine effects or on released factors, both of which have been proven in different experimental models of inflammation and fibrosis [17]. On the other hand, the differentiation capability of PMSCs has been proven in vitro, but it is more questioned in vivo [2].

Studies on PMSCs are exponentially growing, but are still in their early stages. Thus, in order to envision clinical applications, a few unresolved issues need to be considered, some of which are common to MSCs from other sources. For example, MSC heterogeneity and impact of culture conditions on cell characteristics and potentiality is a big issue in the stem cell field. There is a need to have specific markers, which can unambiguously define phenotype and mechanism of action. Other issues that need to be addressed are associated to the fact that the placenta is a recent MSC source, thus the standardization of isolation procedures, safety and stability, tumorigenicity, and efficacy assessments based on studies in different animal models, are still important concerns, and these will be discussed in the following sections.

3.2 Standardization of the isolation procedure

The isolation of MSCs from different placental regions employs either explant culture [2] or enzymatic digestion with various enzymes, such as dispase and collagenase, in combination with DNase. MSCs possess characteristic spindle, fibroblast-like shape in culture and the classical phenotype, also reported for MSCs from other sources, as defined at the First International Workshop on PMSCs, namely expression of CD90, CD105 and CD73, lack of CD45, CD34, CD14, and HLA-DR.

However, PMSCs (like MSCs from other tissues) are highly heterogeneous, and include different populations and different subtypes [2,18]. The differences in phenotype and properties between MSCs isolated from different sources are currently being investigated, but for human term placenta we should also consider that these characteristics may vary between regions of the same organ [10,19]. This should be taken into account when investigating the effects of culture conditions on phenotype and safety. Furthermore, as previously demonstrated, the culture of placenta-derived cells could carry maternal contamination. This constitutes another important issue to be considered, which requires very sensitive tests to determine and/or control fetal or maternal origin after cell culture and expansion [20,21].

3.3 Safety of PMSCs

MSCs are considered as an advanced therapy medicinal product, which include medical products based on genes (gene therapy), cells (cell therapy) and tissues (tissue engineering), and thus are subjected to the same regulation as conventional chemical pharmaceutical drugs.

Indeed, translating basic stem cell research into routine therapies is a complex, multi-step process. The challenge is to manage the expected therapeutic benefits with the potential risks while complying with existing guidelines and regulations. Undoubtedly, the most important point to consider is the safety of these cell products.

Currently, the clinical trials website (www.clinicaltrials.gov) reports 15 trials based on the use of ‘cells derived from placenta’ including those with unknown status. Within the eight completed clinical trials, two were carried out by academic groups and six by private companies. Results obtained from the clinical trial in completed studies have been published. Unfortunately, no details are provided regarding cell manufacturing, including the development of a multi-step controlled and validated process, neither about the quality control approach which should be appropriately designed during and after manufacturing operations [22,23]. This example demonstrates how the safety of some cell products can be overshadowed with respect to their therapeutic efficacy. As previously mentioned, risks and safety should be the main focus in stem cell research and cell therapy today, and this applies also for PMSCs. Therefore, what are the crucial aspects to be considered in order to ensure a safe PMSC product, in addition to improving standardized isolation protocols, as discussed above? We believe the following points should be of interest for the future of PMSCs. First, the purity of the cell product should be determined. This should consider the possible fetal/maternal contamination and also negative clinical effects resulting from engraftment of undesired cell types into an ectopic tissue. A combination of validated analytical methods should define the acceptable percentage of contaminants cells a priori. Second, the tumorigenic potential should surely be considered as a safety evaluation. It is well established that genetic changes can occur during cell culture. Stem cells in particular accumulate chromosomal aberrations, especially at high passages [24], which possibly lead to malignant transformation. Can a specific culture condition, a supplement, cytokine, or xenobiotic-free medium modify and compromise cell stability? These modifications should be thoroughly characterized and the risks assessed before any clinical application. For example, in an allogeneic setting (as is the case for PMSCs), the need for extensive cell quantity expansion in order to obtain enough cells to treat several patients increases the risk of accumulating genetic and epigenetic abnormalities. MSC expansion is associated with a systematic modulation of the epigenetic chromatin structure at specific sites in the genome, but the precise regulation of this process is yet unknown [9]. Finally, senescence should also be taken into account. Cells in culture undergo replicative senescence but chromosomal aberrations are not always detected simply by karyotyping or by single nucleotide polymorphism-microarrays. At early passages, the fibroblastoid colony forming unit frequency and the differentiation potential of MSCs decline significantly, indicating that many cells within the MSC compartment have a restricted proliferative capacity [24].

3.4 PMSC efficacy

The clinical trials mentioned above are Phase I and II, thus they are mostly aimed at safety evaluation. In regard to efficacy, lessons can be learned from preclinical studies, which have shown great promise. PMSCs have shown to be able to restore the impaired or even lost function and delay the degenerative process in models of debilitating neurological diseases such as Parkinson’s disease [25], Alzheimer’s disease [26,27] and multiple sclerosis [28]. Furthermore, other studies employing PMSCs and their derivatives in animal models of lung disease have demonstrated reduced inflammation and fibrosis [15,29,30]. Other groups have shown disease attenuation after treatment with PMSCs in animal models of myocardial infarction [31,32], and also in autoimmune diseases such as rheumatoid arthritis [13]. Furthermore, preclinical studies have shown that PMSCs may also be a helpful in liver diseases, as suggested by their potential to differentiate toward functional hepatocyte-like cells in vitro [33], and also owing to their immune modulatory properties. Importantly, preclinical studies have and will continue to pave the way for further studies focusing on the mechanism of actions underlying disease amelioration.

4. Concluding remarks

In conclusion, there is plenty of evidence demonstrating efficacy of placenta-derived cells or CM derived from these cells in diseases with underlying inflammatory and fibrotic abnormalities [15,17,30]. Attempts to improve and standardize methods have been reported, however much remains to be defined. It is unclear whether the current knowledge is sufficient to adequately assess the safety of new cell therapies in a comprehensive manner. The current concept of safety is not unambiguous, but we are surely evolving. The safety of a product will go hand in hand with the increase of knowledge on these cells. Importantly, cell biologists, therapy providers, clinicians and regulatory agencies need to interact to define the acceptable risk associated with PMSCs treatment, and to establish a framework for accurate risk assessment.

An obvious question arises regarding the need for an alternative source of MSCs, such as placenta, and if placental cells could target specific diseases compared to MSCs from other sources. Indeed, placenta as a source of other stem cells, besides MSCs, could reserve some interesting promises, while placenta as an MSC source is fascinating if we foresee to produce off-the-shelf products from biological waste. If the safety and efficacy of PMSCs can be proven comparable, or better than, MSC from other sources, then placenta could become an important, unforeseen cell source, not only of hematopoietic stem cells (as demonstrated by placental cord blood), but also of MSCs.

Declaration for interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.