Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties

In recent years, divergent populations of postnatal mesenchymal stem (stromal) cells (MSCs) have been identified in various tissues, including umbilical cord, bone marrow, adipose and oral tissues [1], and have been extensively studied for their safety and efficacy for numerous clinical applications [2, 3]. MSCs attract worldwide interest for their multilineage differentiation potential into tissue-forming cells having the ability to support repair processes in damaged sites, but more importantly for the paracrine-mediated regenerative effects that they exert through the release of a plethora of trophic and anti-inflammatory/immunosuppressive factors in their secretome, with potential to modulate the host immune responses [4, 5].

Although more than 180 MSC-related clinical trials are currently registered in a database compiled by the US National Institutes of Health (www.clinicaltrials.gov), considerable variation in the mode and procedures of preparing MSCs for clinical use is evident, thus unfolding the urgent and unmet clinical need for establishing universally accepted materials and standard operating procedures (SOPs) for safe, reproducible and economic isolation and large-scale expansion of MSCs at a clinical-grade level. MSCs are currently classified as advanced therapy medicinal products (ATMPs), and therefore are subject to Regulation EC1397/2007 of the European Commission [6]. A recent definition also designates MSCs as cell-based medicinal products (CBMPs); that is, medicinal products presented as having properties for, or used in or administered to, human beings with a view to treating, preventing or diagnosing a disease in which the pharmacological, immunological or metabolic actions are carried out by cells or tissues [7]. These products have to be prepared in compliance with good manufacturing practices (cGMP) that ensure consistent production and controlled quality standards appropriate to their intended use, as described in EU Regulation 2003/94/EC [8]. Such cGMP practices include, among others, several parameters related to MSC isolation and cultivation processes. To specify procedures for proper handling under cGMP conditions, current research focuses on evaluating the effects that expansion procedures might have on the MSC “stemness” characteristics, their phenotypic and genetic stability, the efficacy in regenerating the target tissues and the permitted population doublings (PDs) before senescence emerges to finally establish reliable characterization methodologies for the accurate assessment of each of these parameters [9].

Considerable effort has been devoted to optimizing standard culture protocols. In particular, the use of animal serum as supplementation for conventional culture media is fraught with problems. Although still widely used, its application raises serious concerns related to the highly variable and often unknown composition of each batch, the immunological risks associated with serum proteins and, most importantly, the potential transmission of animal diseases to humans [10]. Serum components in MSC cultures are crucial for effective cell expansion [11] and efforts to replace commercially available bovine or calf serum with cGMP alternatives, such as autologous or allogeneic human sera or human platelet lysate (HPL), have encountered technical difficulties in achieving large-scale expansion of MSCs, and also in meeting the laboratory requirements of these supplements in large amounts [12]. Most recently, the development of cGMP culture systems, free of any animal derivatives (i.e., serum/xeno free), has been achieved by leading life science companies aiming to provide homogeneous, well-defined MSC populations with enhanced “stemness” characteristics, minimized variability and closely controlled MSC functions, meeting the EU regulatory requirements [13].

Another important element for successful implementation of MSC-based therapies is related to selection of the most suitable source to isolate the MSCs, tailored to the intended clinical application [14]. In the field of dental research, various oral MSC sources have been proposed as highly promising for tissue engineering (TE) applications in the orofacial region. Among these, the alveolar crest has been suggested as an advantageous source for alveolar bone marrow-derived MSCs (aBMMSCs) used in maxillofacial bone regeneration [15], as compared to the iliac crest-derived BMMSCs, considered so far to be the “gold standard”. aBMMSCs have been shown to proliferate faster, demonstrate delayed senescence in vitro and form bone in greater amounts in vivo, compared to the iliac crest-derived BMMSCs of the same individuals [16]. An additional benefit of aBMMSCs is the minimally invasive surgical technique required to obtain the osseous biopsies during routine dental procedures (i.e., tooth extraction, implant placement).

Another promising oral MSC source of great importance in regenerative dentistry is the pulp of the adult teeth (dental pulp stem cells (DPSCs)). These cells have been studied extensively for in-vitro multilineage differentiation potential toward osteo/odontogenic, adipogenic, chondrogenic, neurogenic, angiogenic and myogenic lineages [14, 17], while in-vivo studies confirm their ability to reconstitute functional dentin/pulp complexes [18] and other tissues including bone [19], cementum [20], blood vessels [21] and neural tissues [22]. A growing number of “proof of concept” studies validate DPSCs as a very effective MSC source for dental [23] and nondental biomedical applications [24].

In the context of establishing standardized conditions for safe and efficient clinical-grade expansion of oral MSCs, bridging the gap between research models and clinical applications in the orofacial region, the present study aimed to identify the critical “stemness” properties influenced by the culture “micro-milieu” that might have an impact on the MSC clinical performance. The potential of two commercially available serum/xeno-free, cGMP culture systems to maintain the growth and functional properties of two types of oral MSCs (DPSCs and aBMMSCs) was investigated in comparison with a widely and longtime used conventional fetal bovine serum (FBS)-based approach. The growth rates, differentiation potentials and morphological, immunophenotypic and senescence characteristics of MSCs were determined for both cell types after prolonged expansion. The results of this study contribute to the development of qualified protocols and SOPs for clinical-grade expansion of oral MSCs, as a key milestone achievement for translation of research findings to clinical settings.

The discovery of the unique biological properties of human MSCs has generated a plethora of clinical trials designed to test their safety and efficacy for the treatment of various pathologies. In dental research, a number of case reports have been conducted to test the regenerative potential of oral MSCs in orofacial osseous [30] and pulp regeneration [31], whereas randomized controlled clinical trials (RCTs) are quite sparse especially in humans [32, 33]. An electronic search in the ClinicalTrials.gov database (accessed July 2017) implemented by the US National Institutes of Health revealed six ongoing clinical trials administering oral MSCs in various applications, including periodontal and peri-implant therapies, cleft lip and palate management and revitalization of immature permanent teeth with necrotic pulp [34]. Nevertheless, the clinical application of MSC-based therapies has been constrained, among other reasons, by difficulties in the ex-vivo expansion of clinical-grade MSCs under good manufacturing practice (GMP) conditions, in compliance with EU regulations [6, 8]. To our knowledge, this is the first comprehensive study providing extensive characterization of oral MSCs (DPSCs and aBMMSCs) after prolonged expansion with two proprietary culture systems fulfilling cGMP requirements, as compared with a conventional serum-based medium.

It is widely accepted that differing culture conditions may affect MSC properties [3]. Since preparation of high-quality MSCs is required for any safe and efficient cell therapy treatment, considerable efforts have been made to evaluate the consequences of cultivation processes on stem cell behavior. It has been reported that prolonged culture of bone marrow stromal cells correlates with various functional changes, including reduced proliferation, ultimately producing replicative senescence [9] and associated molecular changes [35], together with reduced [36] or shifting multilineage differentiation potential from highly osteogenic to more adipogenic [37]. However, currently there is not much in-depth knowledge regarding the precise effects of the culture micro-milieu on the “stemness” properties of oral MSCs. The present study has performed a rigorous parallel investigation of two oral MSC populations of different origin (i.e., from adult dental pulp (DPSCs) and alveolar orofacial bones (aBMMSCs)), both recommended for dental tissue and orofacial bone TE, respectively [14, 17, 30, 31]. The extensively used animal serum (bovine or calf) and porcine-derived dissociation agent trypsin, although still tolerated by the regulatory authorities as being used in ongoing clinical trials, are not considered compatible with cGMP practices, while other serum-based cGMP alternatives, including human autologous or allogeneic serum [38] or HPL [39], impose several limitations regarding large-scale cell expansion [40] and reagent availability. Based on these shortcomings, this study sought to trace potential quenching of critical stem cell properties after isolation and subsequent prolonged expansion of oral MSCs under serum/xeno-free, cGMP compared to conventional serum-based culturing conditions.

There is a growing effort by leading life science companies to develop serum/xeno-free media formulations that are in full compliance with GMP requirements, as defined by the EU, in addition to relevant “supportive” reagents, namely cGMP culture vessel coatings, dissociation and cryopreservation reagents, and so forth, in order to fully support clinical-grade production of MSCs for therapeutic use. However, a basic drawback is that the manufacturers do not provide any information on the chemical formulations of these proprietary media, other than certificates on their GMP compliance. On the other hand, there are quite a large number of developed serum-free stem cell expansion media provided by several companies, mainly aiming to replace calf/bovine serum due to its well-known disadvantages. These media are characterized as serum free, but not as cGMP.

There is quite rigorous and well-performed, previously published work on how the chemistry of various “house-made” serum-free media may affect “stemness” properties, such as PDs and phenotype expression of the stem cells. More specifically, several authors have investigated the effects of a wide range of medium supplements (including FGF-2, PDGF-BB, ascorbic acid, EGF) as well as other culture medium ingredients (such as glucose content, addition of l-glutamine vs Glutamax) on “stemness” properties of various MSC types, with variable results [11, 41–45]. However, very often there is a lack of direct comparison between paired samples, thus making it difficult to outweigh the actual effects of switching supplements. Furthermore, these disclosed formulations should ensure the biosafety of the media ingredients, which requires further testing.

In order to address these challenges by “in-house” serum-free media of known consistencies, newly developed proprietary serum/xeno-free, cGMP media have been developed as promising alternatives for the manufacturing of clinical-grade MSCs. Until now, there are sparse comparative studies evaluating these proprietary cGMP media and their impact on “stemness” properties of various types of MSCs [46–49]. Nevertheless, in most of these studies, StemPro (Life Technologies) is evaluated and proposed as a promising alternative to classic serum-containing media. For this reason, the current study elected to use this medium and make direct comparisons of two different oral stem cell populations to other nonoral MSC types. StemMacs, on the other hand, belongs to a new generation of recently developed cGMP culture systems and therefore was also included in this study, since comparisons of more than one serum/xeno-free, cGMP medium with a standard (FBS) serum-based medium documents a more comprehensive characterization of the innate potential of the oral MSCs to support the initial isolation procedures, and their robustness to provide adequate cell numbers over prolonged serum-free expansion.

Research findings of the present study demonstrated similar isolation efficiency under serum-free conditions for both DPSCs and aBMMSCs, as the required time to reach p.1 for each cell type was similar for the three media tested, although the overall time was higher for aBMMSC (approximately 12–14 days) compared to DPSC (approximately 9–11 days) cultures. This could be partially attributed—except from the obvious reason of different tissues of origin—to the younger age of the DPSC donors over the aBMMSC donors, since age is known to significantly affect biological properties such as the proliferation rate [50] and to be associated with epigenetic alterations, telomere attrition, accelerated cellular senescence, stem cell exhaustion and altered intercellular communication [51]. In addition, the expanded DPSCs under serum-free conditions produced similar growth patterns during consecutive passaging compared to the serum-based conditions (Fig. 1), whereas StemMacs-expanded aBMMSCs showed a pronounced increase in PDT after middle passages (p.6–7). However, it has to be mentioned that all media were able to achieve cell yields equal to (aBMMSCs) or greater than (DPSCs) 30 million cells—corresponding to a generally recommended clinical dose for intraoral applications—after completion of no more than three (early) passages. Several commercial serum/xeno-free media have recently emerged in the market as promising tools for establishing clinical-grade MSCs with enhanced “stemness” characteristics. A few studies have investigated various serum-free proprietary media as promising alternatives to in-house-developed serum-free media [46–49] and have shown that these products are able to isolate and expand various MSC populations, adhering to the minimal criteria set by the International Society of Cellular Therapy (ISCT) and often providing an enhanced performance compared with the conventional media [52]. However, some of these studies are not standardized for several parameters, such as proliferation rates or variability in the differentiation potential of the cells among media. The present study highlighted the importance of thoroughly exploring MSC properties of different cell populations grown in various serum-free media, as the highly proliferative DPSCs were not severely inhibited—at least regarding cell growth—under both serum-free media, while the less proliferative aBMMSCs showed a dramatic elimination of proliferation dynamics after prolonged expansion with one of these media (StemMacs). As new generations of serum-free media emerge from various companies, these data may be significant in addressing particular challenges related to the preparation of oral MSCs for therapeutic use.

Notably, growth kinetics were not fully accordant with morphological differences and senescence data, implying that multiple criteria have to be considered to fully characterize “stemness” of oral MSCs. DPSCs expanded under serum-free conditions showed similar growth patterns to serum-expanded cells (Fig. 1), but exhibited increased SA-β-gal positivity (especially at late passages) (Fig. 4), accompanied by a generally elevated cell size distribution and granularity (Fig. 2). In contrast, aBMMSCs contained significantly more SA-β-gal-positive cells compared to DPSC cultures, which was more pronounced in the StemMacs-expanded cells, in accordance with the growth and morphological data (Figs. 1, 2 and 4). The limitation of cell division in cultures is a known phenomenon for all somatic cells unless the cells are immortalized [53]; after approximately 40–100 divisions, depending on cell type and culture conditions, the proliferation rates decay, with eventual domination by the senescent state [46]. Nevertheless, it should be noted that, despite increased numbers of SA-β-gal-positive cells, the reduction in the mean telomere length with increasing passage number, although evident, remained minor, failing to reach statistical significance for all of the cells/media studied (Fig. 5). Telomere length is a primary indicator of MSC replicative (telomere-associated) senescence [29, 54] characterized by progressive loss of the telomeric TTAGGG repeats [55]. The results of this study suggest that variations in growth kinetics among different oral MSC types and/or expansion media are not directly attributable to accelerated telomere loss. It has been contended that SA-β-gal activity, although associated, is neither causative nor specific for senescence [54, 56, 57]. Instead, it should be interpreted in combination with other biomarkers [56, 58]. However, given the expression of β-galactosidase has been mostly associated with a nonproliferative state, the increased proportion of such cells may be implicated in the disparate growth characteristics observed for different oral MSCs and/or expansion systems. In line with present findings, a previous study [59] showed no significant shortening (<1 kbp) in mean telomere length of aBMMSCs at early (p.1–4; PD < 25) and middle (p.5–8; PD < 50) passages, while at the same time significantly greater proportions of SA-β-gal-positive cells became evident over time. It has also been shown that significant heterogeneity existed among different DPSC subpopulations, with those capable of exceeding 80 PDs before senescence occurred possessing longer telomeres (18.9 kbp) than the less proliferative populations (<40 PDs) which had much shorter telomeres (5–13 kbp) [60]. Although there is a general recommendation that expansion of bone marrow MSC preparations for clinical use should be limited to 4–7 PDs [61], the current study, in addition to previous work, aimed to look into the replicative potential of these MSC populations and set the limits and standards for effective cell expansion in order to attain the desired cell numbers before senescence occurs. Indeed, activation of senescence has been demonstrated to produce morphologic and functional changes such as alterations in nuclear structure, protein processing, gene expression and cell metabolism [62] that might compromise the clinical outcome. In addition, senescence has been associated with the so-called “senescence-associated secretory phenotype” (SASP), which might be detrimental to regeneration as the senescent cells may release numerous cytokines, growth factors, proteases and extracellular matrix components inducing a state of chronic inflammation [63]. It has been further shown that decreased expression of cytokine and chemokine receptors in aged BMMSC populations compromises their protective role by limiting potential for activation and mobilization to the injury site [64].

Of interest was that oral MSCs expanded with serum-free media exhibited a very homogeneous morphology comprising well-aligned spindle-shaped cells (Fig. 2). This finding is in accordance with previous data [65] and also with the specifications of the manufacturers regarding the selective isolation of homogeneous MSC populations. However, knowing the exact composition of these media is essential to justify such postulations. In contrast, serum-expanded oral MSCs (both DPSCs and aBMMSCs) showed higher population heterogeneity comprising cells of various shapes and sizes. Heterogeneity of the cells isolated by the enzymatic dissociation method that are “loosely” termed “MSCs” [66] has been related to cultures containing cells at different stages of developmental commitment, including stem cells, rapidly dividing transit amplifying (TA) cells that become progressively lineage restricted while dividing [60].

The present study illustrated the variable expression patterns of MSC markers as important hallmarks of “stemness”. Well-recognized MSC markers such as CD90 and CD73 in addition to CD81 and CD49f were stably and abundantly expressed in both cell types under serum-based as well as serum-free conditions up to late passages (Fig. 3a–f), thus being totally unaffected by any of the observed morphological, growth, senescence or differentiation changes of the cells. In contrast, other MSC markers such as CD105, CD146, STRO-1 and the embryonic markers SSEA-1 and SSEA-4 were more sensitive in “following” such morphological/biochemical alterations after prolonged passaging, and showed more pronounced downregulation during serum-free-based (all markers except SSEA-1 reduced at similar levels) as compared to serum-based expansion of both cell types (Fig. 3a–f; Additional file 1). These findings contrast with a recent study [49] that demonstrated a very low and unaltered expression of CD105 and SSEA-4 with prolonged passaging in human DPSCs, while other studies confirmed downregulation of SSEA-4 during differentiation in human MSCs [59]. SSEA-4 has been characterized as a classical embryonic stem cell marker used in isolating adult MSCs with enhanced multilineage differentiation potential [67] and capacity for forming organized bone tissue in vivo [68].

The present study also confirmed the upregulation of the initially minimally expressed CD34 (<10%) after prolonged expansion; a consistently apparent donor-related finding both in DPSC and aBMMSC cultures irrespective of the culture conditions. A recent concise review [69] reports that although CD34 is associated with the selection of hematopoietic stem cells from bone marrow transplants, it is also expressed by MSCs, as well as by other nonhematopoietic cell types, indicating a distinct cell subset with enhanced progenitor activity. Therefore, the ISCT consensus of CD34 as a negative MSC marker has not been verified by some reports, which demonstrate that CD34 is an important marker in early, freshly isolated MSCs, associated with high colony forming efficiency and prolonged proliferative capacity, but whose expression gradually diminishes with passaging [70]. Current results showed increasing CD34 expression with passaging, in line with a previous report [70] which found an increase of baseline expression of CD34 in adipose MSCs at 15 days followed by downregulation at 30 days, while CD90 remained consistently highly expressed, as in the present study. Increased CD34 expression was related to the acquirement of proendothelial characteristics, since CD34 was also highly expressed in endothelial progenitors [71]. Investigation of the angiogenic potential of oral MSCs after prolonged culture could clarify this position.

Present results also highlighted that expansion with both serum-free systems resulted, even at very early passages (p.2–3), in a “predisposition” toward an increased baseline expression of osteogenic markers, with parallel complete elimination of chondrogenic (the late marker ACAN, counteracted by overexpression of the transcription factor SOX-9) and adipogenic markers (both the early and late markers PPAR-γ and LPL, respectively), whereas serum-based expansion comparatively produced only minor changes in osteogenic markers and less prominent changes in adipogenic and chondrogenic markers (Fig. 6a–l). Evidence indicates that osteogenesis and adipogenesis are inversely regulated by their master transcription factors, Runx2 and PPAR-γ, respectively [72]. Chondrogenesis and osteogenesis share common transcriptional regulatory pathways via activation of Runx2/Cbfa1, but deviate in several other control points. In particular, chondrogenesis is strongly regulated by SOX-9 that remains continuously present from early stages of MSC condensation to become chondrocytes until late stages of chondrocyte hypertrophy [72]. SOX-9 regulates expression of cartilage extracellular matrix proteins, such as Col2A1 and ACAN [73]. It is therefore remarkable that SOX-9 upregulation was accompanied by elimination of expression of ACAN. A possible explanation could be that this effect is due to a negative transcription regulation, and warrants further investigation.

We further evaluated the impact of this “osteogenic predisposition” of serum-free expanded oral MSCs on their multilineage differentiation potential toward osteogenic and chondrogenic phenotypes. Notably, serum-expanded DPSCs retained a diminishing osteogenic differentiation potential with passaging, while serum-expanded aBMMSCs lost this potential by reaching the middle passages (Fig. 7a–f). This finding concurs with previous reports of reduced multipotency of DPSCs [74] and BMMSCs from other sources [15, 75], and is also consistent with the mineralization potential analysis data (Fig. 7g, h). On the other hand, StemMacs-expanded DPSCs and aBMMSCs showed a diminishing expression of most initially highly expressed osteogenic markers during osteogenic differentiation. However, the mineralization potential increased in DPSCs with passaging, and in the case of aBMMSCs the initially very high mineralization potential was eliminated after reaching middle passages. The conclusion that can be drawn for this part is that the overexpression (compared to the serum-based conventional conditions) of osteogenic markers in StemMacs-expanded DPSCs is related to an increasing in-vitro mineralization potential with passaging, while in the case of aBMMSCs this advantage over serum-based expansion could be only observed at early passages. Finally, StemPro-expanded DPSCs showed further increase of most of the initially highly expressed osteogenic markers (at least at early to middle passages), which was also partially evident (only for ALP) in the StemPro-expanded aBMMSCs. This increase was attenuated with passaging, however, and this was also true for the in-vitro mineralization potential of both cell types, which decreased for the DPSCs and was eliminated for the aBMMSCs. It is therefore obvious that the overexpression of osteogenic markers in StemPro-expanded DPSCs and aBMMSCs is not related to a sustainable in-vitro mineralization potential with passaging, as compared to the serum-based conventional conditions. Overall these results highlight the need for carefully selecting cell populations and media to meet the needs of a particular clinical situation, taking into account that these cells can be actually “trusted” to maintain the highest degree of osteogenic potential only at early passages; the latter statement referring to both serum-based and serum-free conditions.

Finally, chondrogenic marker expression increased with passaging in serum-expanded DPSCs but decreased in serum-expanded aBMMSCs. Serum-free expansion was associated with a dramatic decrease (in StemPro-expanded DPSCs and aBMMSCs) or a complete elimination (in StemMacs-expanded cells) of the baseline chondrogenic marker expression, further confirming that there seems to exist a “selection” of the serum-free-expanded populations toward osteogenic progenitors. These data, however, require further functional confirmatory assays before incorporating them into standardized protocols.

The present study provides novel insights into the critical parameters that define oral MSC “stemness” mostly influenced by the culture “micro-milieu” and have the potential to predict the isolation efficiency, growth and functional properties of MSCs before clinical application. It is evident that several biological endpoints, including morphological, growth, senescence, immunophenotypic, gene expression and differentiation characteristics, have to be comprehensively considered to assess maintenance of oral MSC “stemness”. It also becomes apparent that the establishment of new protocols of clinical-grade, cGMP expansion of MSCs is not trivial, and the feasibility of cGMP production of MSC preparations that meet the regulatory criteria is often ambivalent. Other parameters, such as the replacement of recombinant enzymes (collagenase and dispase) with cGMP reagents (e.g., the recently proposed highly purified GMP-grade enzyme blend named liberase; Roche Custom Biotech, Mannheim, Germany) or even complete restraint for enzymes (i.e., using the explant culture method [25]), also require consideration. The results of this study clearly show the feasibility of isolating and expanding DPSCs and aBMMSCs using cGMP, serum/xeno-free culture systems without compromising basic “stemness” properties at least in early (for aBMMSCs) or up to middle (for DPSCs) passages. The high baseline expression of osteogenic markers in serum-free-expanded MSCs could be of advantage in bone and/or other mineralized tissue (e.g., dentin) regeneration applications. Finally, typical MSC markers such as CD90 and CD73 appear not to be specific in indicating maintenance of “stemness” and should therefore be supplemented by multiparametric immunophenotyping, including at least CD105, CD146, SSEA-1, SSEA-4 or other embryonic SC markers. Specific additional challenges, such as the extremely high cost of these new cGMP media, also need to be taken into consideration for large-scale clinical applications. Finally, as different media produce MSCs with differential “stemness” properties, the most suitable protocol for clinical-grade MSC expansion should be carefully selected and validated in preclinical animal models to fit the regenerative requirements of the target tissues.