Abstract

Introduction

Ability to isolate and establish pluripotent or multipotent stem/precursor population is a major objective for the biotechnological industry and clinical translation of regenerative medicine. A major impediment for using stem cells in a clinical setup is poor availability of cells, especially those obtained with noninvasive procedures without raising much ethical issues. These limitations greatly restrict the usage of stem cells in clinics, disabling treatments of many degenerative diseases. This lacuna can be filled if any tissue-specific cells can be verifiably demonstrated to possess pluripotent or multipotent capacity. This may elevate the hope to find a well-suited stem cell-like cell line that can serve as an autologous, noncontroversial, and renewable source for cell therapy without ethical and immunological concerns, which are usually associated with embryonic stem cells (ESCs).

Numerous gene and protein expression criteria have been set for recognizing a cell as pluripotent. Microarray analyses have demonstrated a set of various transcripts that are associated with stemness as in the case of ESCs [1]. Notably, it has been demonstrated by Yamanaka and colleague that the combinations of four major transcription factors, Oct-3/4, Nanog, Sox-2, and Klf-4, is indispensable for the cells to maintain pluripotency as in the case of ESCs [2,3]. However, there are some other reports which indicate that expressions of not all the four genes are essential to maintain the stemness. According to NIH and ISSCR guidelines, teratoma formation is one of the major criteria for classifying a cell to be pluripotent [4]. Apart from this, the presence of alkaline phosphatase (ALP) is another reliable property shown for pluripotent cells such as ESCs [1,5].

In principle, a cell that is able to differentiate into cell types of all three germinal layers is considered pluripotent. On the other hand, a multipotent cell can give rise to cells originating from the same germ layer [6]. A classic example of a stem cell with pluripotent and multipotent potential is mesenchymal stem cells (MSC). MSCs can differentiate into various cell types. Depending on isolation procedure and tissue source, both pluripotent and multipotent type of stem cells can be isolated [7]. Mesenchymal cells display fibroblastic cell morphology and express vimentin protein as a characteristic marker along with high Oct-3/4, Nanog, and Sox-2 (ONS) expression [8]. Bone marrow MSC (BMSC) isolated from crude bone marrow are reported to possess pluripotent gene expression and also show tri-lineage differentiation [9–12]. Therefore, it appears that BMSC can serve as a model that is used for screening various cell types for their degree of stemness. Hence, cells which portray specific features that are in accordance with the parameters mentioned earlier can be designated as pluripotent cells.

In a very recent report, Wang et al. exemplify that ONS genes are the core regulators of pluripotency. This group showed that Oct4 regulates and interacts with the BMP4 pathway to specify four developmental fates. High levels of Oct4 enable self-renewal in the absence of BMP4; Nanog represses embryonic ectoderm differentiation but has little effect on other lineages, whereas Sox-2 is redundant and represses mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates [13]. Numerous cell types isolated from tissues and organs such as heart, kidney, liver, lungs, brain, pancreas, spleen, muscles, adipose tissue, dental pulp, placenta, and amnion are now being tested on the parameters mentioned earlier with an aim of generating patient-specific pluripotent stem cell lines for treating various incurable degenerative diseases.

With numerous published reports, it becomes a general belief that pluripotency, stemness, and differentiation potential into trigerminal lineages are associated with high levels of ONS genes, but this does not explain the fact that many low ONS expressing cells can also demonstrate multilineage differentiation potential, high ALP staining, and a teratoma-like structure formation and, hence, generate valid questions. (1) What should be the threshold of ONS gene expression for any cell to be pluripotent? (2) Is pluripotency really a function of high ONS gene expression or can basal level expressing cells also show lower but sustained pluripotency? Attempts are made in the present study to answer these questions scientifically using NIH3T3 as a model and are compared with high ONS expressing cells.

NIH3T3 cells, isolated from Swiss albino mice [14], are not known as stem cells due to the reduced expression of pluripotency-associated genes despite being embryonic in nature. These cells have been extensively used as a model system for various molecular, cellular, and toxicological studies [15–18] and are considered differentiated dermal fibroblast cells. Many groups used NIH3T3 as control cells that fail to differentiate [19–21]. However, their differentiation into adipocytes, myocytes, and neural cell types has been reported either after transforming genetically or if differentiated under defined media conditions [19–21]. Piestun et al. reported that NIH3T3 cells after being transfected with Nanog show induced pluripotency and expressed markers that are specific to various differentiating cell types [22]. In 2006, Yamanaka and colleagues also compared pluripotent ESCs and untransformed NIH3T3 by a microarray analysis and concluded that although these cells did not express pluripotency genes, especially the four crucial ones, that is, Oct3/4, Nanog, Sox-2, and Klf4; they expressed c-Myc [3]. However, this group neither examined nor commented on the multipotent nature of these cells. Later, in 2008 and 2009, Deng and colleagues published two reports demonstrating differentiation of untransformed NIH3T3 cells into oesteocytes and neuronal cell types in bone and spinal cord injury mouse models [1,23,24]. Two other groups also reported that NIH3T3 can give rise to neural cells and oesteoblasts, respectively [21,25]. Recently, our group also reported pancreatic islet cell differentiation of untransformed NIH3T3 with appropriate induction [26].

To answer these questions and to understand whether pluripotency can also be associated with basal ONS gene expression, we carried out a comparative study of basal ONS expressing NIH3T3 cells with pluripotent mouse BMSC (mBMSC) and mouse ESC (mESC). We carefully examined NIH3T3 cells for pluripotent and multipotent stem cell specific markers and compared them with those of mBMSC and mESC with regard to morphological features, expression profile of stemness related genes, ability to undergo multilineage differentiation, and in vivo teratoma formation.

Materials and Methods

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Abstract

1. Introduction

Stem cells are immature tissue precursor cells which are able to self-renew and differentiate into multiple cell lineages [1, 2]. Mesenchymal stem cells (MSCs), also known as multipotent mesenchymal stromal cells, are self-renewing cells which can be found in almost all postnatal organs and tissues [3, 4]. MSCs have received wider attention because they can be easily isolated from a small aspirate of bone marrow or adipose tissue and can be expanded to clinical scales in in vitro condition. Other than these MSCs offer several other advantages like long-term storage without major loss of potency and no adverse reactions to allogeneic MSCs transplant [5].

In 1976 Friedenstein et al. firstly described a method for MSCs (referred as “stromal cells”) isolation from whole bone marrow aspirates based on differential adhesion properties. They suggested that these cells are adherent, clonogenic, nonphagocytic, and fibroblastic in nature, with the ability to give rise to colony forming units-fibroblastic (CFU-F) [6]. In late 1980s Owen and Friedenstein reported heterogeneity of the bone marrow stromal cells for the first time [7, 8]. Bone marrow stromal cells were further characterized and named mesenchymal stem cell to describe the subtype of marrow stromal cells involved in the process of mesengenesis [9, 10]. Shortly after these discoveries researchers started to explore the therapeutic application of MSCs [11], since then no adverse effect of MSC transplantation has been reported. In this paper we tried to compile recent advances in the MSCs research and its medical implications.

2. Immunophenotype of MSC

The identification of MSCs with the use of specific markers remains elusive. There is no single surface marker, but rather a panel of surface markers which define Human MSCs (hMSCs), derived from fresh tissues or cryopreserved samples. As per the international society for cellular therapy guidelines, MSCs must express CD105 (SH2), CD73 (SH3/4), and CD90 and must be negative for surface markers CD34, CD45, CD14, CD79α or CD19, and HLA-DR [9]. hMSCs are also negative for several other antigens like CD4, CD8, CD11a, CD14, CD15, CD16, CD25, CD31, CD33, CD49b, CD49d, CD49f, CD50, CD62E, CD62L, CD62P, CD80, CD86, CD106 (vascular cell adhesion molecule [VCAM]-1), CD117, cadherin V, and glycophorin A. On the other hand, hMSCs are positive for CD10, CD13, CD29 (b1-integrin), CD44, CD49e (a5-integrin), CD54 (intercellular adhesion molecule [ICAM]-1), CD58, CD71, CD146, CD166 (activated leukocyte cell adhesion molecule [ALCAM]), CD271, vimentin, cytokeratin (CK) 8, CK-18, nestin, and von Willebrand factor [5, 12, 13]. Tissue specific expression of surface marker is well noted such as only adipose tissue-derived MSCs express high levels of CD34 [14] and bone-marrow-derived MSCs, but not placenta derived MSCs, express CD271 [15]. Detailed phenotypic expression of surface markers is reviewed elsewhere [16].

3. Differentiation Potential of MSC

Other than surface markers MSCs must have ability to adhere to plastic and differentiate into osteoblasts, adipocytes, and chondroblasts under in vitro condition [9]. Differentiation is regulated by genetic events, involving transcription factors. Differentiation to a particular phenotype pathway can be controlled by some regulatory genes which can induce progenitor cells’ differentiation to a specific lineage. Besides growth factors and induction chemicals, a microenvironment built with biomaterial scaffolds can also provide MSCs with appropriate proliferation and differentiation conditions [17]. Even though MSCs can differentiate into a number of tissues in vitro, the resulting cell population does not mimic the targeted tissues entirely in their biochemical and biomechanical properties [18].

4. Migration and Homing

The physical niche and migration signals of MSCs provide invaluable information about their role and interactions within the tissue. Bone-marrow-derived MSCs received more attention from researchers in hopes of revealing clues about their therapeutic activity. During in vivo condition, it is difficult to locate MSCs’ niche. Moreover due to the lack of any specific MSCs marker and difficulties in probing marrow cavities, it is very difficult to track dynamic movement of MSC. Most researchers use genetic markers such as Y-chromosome, when male cells are introduced into females or fluorescent protein reporter genes but these methods do not resolve the dynamics of cellular and temporal responses and are not quantitative [5]. Noninvasive in vivo imaging accomplished by using bioluminescence imaging (BLI) can be a possible solution. The main advantage of BLI is that even at very low levels of signal, as few as 100 cells can be detected in vivo [38, 39]. Significant advances have been made in this field but still MSCs migration to tissue niche is illusive.

MSCs migration to injured tissues has been reported in radiation-induced multiorgan failure, ischemic brain injury, myocardial infarction, and acute renal failure [40], but the mechanisms that regulate the MSCs migration to the injured tissues are still unknown. Human MSCs express different combinations of the chemokine receptors CCR1, CCR4, CCR7, CCR9, CCR10, CXCR1, CXCR3, CXCR4, CXCR5, and CX3CR1 [41]. The chemokine(s) that control MSCs trafficking are still unknown; while to date, 39 chemokines have been identified with different functions controlling the traffic of hematopoietic cells, in particular leukocytes [41]. Among these chemokines, stromal cell derived factor-1 (SDF-1) is relatively well studied for MSCs migration.

SDF-1-induced cell migration is mediated by its receptor, CXCR4, which is broadly expressed in cells of the immune system and in the central nervous system (CNS). The role of SDF-1 as an important mediator of stromal progenitor migration to injured tissue has been reported in vivo using a rat model of myocardial infarction [42, 43]. Hiasa et al., 2004 reported that the overexpression of human SDF-1 in the ischemic muscle induced the mobilization of endothelial progenitor cells and improved myocardial healing. Studies also demonstrated that after myocardial infarction the levels of SDF-1 are increased in infarcted tissue and this increase correlates with the number of MSCs that home into the heart [42, 43]. On the other hand, study by Ip et al., 2007 suggested that MSCs use integrin β1 and not CXCR4 for their myocardial migration [44]. Moreover, in regenerating skeletal tissues, the MSCs homing may be improved with growth factor delivery, as combined MSCs and erythropoietin infusion gave better results in limb ischemia treatment [45]. Bioactive lipid lysophosphatidic acid (LPA) plays a principal role in the migration of human lung resident MSCs through a signaling pathway involving LPA1-induced beta-catenin activation [46]. Anti-inflammatory environment is more accommodating to the therapeutic hMSCs than a proinflammatory environment [47].

Crossing of the endothelial barrier is another critical step for the tissue migration of circulating cells. Similarly to leukocytes, MSCs adhesion to the endothelial cells represents a critical step and a restricted set of molecules such as selectin-P, integrin β1, and VCAM-1 and seems to play critical roles in this interaction [48]. The in vivo homing potential of MSCs circulating in the bloodstream to the sites of injury/inflammation can be regulated by adhesion of MSCs to endothelium, achieved by pretreatment of endothelial cells with some proapoptotic agents, angiogenic and inflammatory cytokines, and growth factors, such as interleukin (IL)-8, neurotrophin-3, TGF-β, IL-1β, TNF-α, platelet-derived growth factor, EGF, and SDF-1 [12]. Further studies into understanding the molecular mechanism behind migration and homing will provide an impetus to the use of MSCs for therapeutic purpose.

5. Mechanism of Action/Mode of Action

The mechanism by which MSCs exert their antiproliferative effect have still to be fully elucidated, although several mechanisms and molecules have been proposed that are likely to act in concert and/or in alternate fashion depending on the environment conditions to which MSCs are exposed. Several studies have shown that MSCs are capable of replacing damaged tissues in vivo [49, 50]. Multiple tissue engineering approaches have also been reported where undifferentiated or predifferentiated MSCs were delivered with or without help of biomaterial [49, 50]. MSCs have shown promise in replacing various tissues including cartilage, bone, tendon, vasculature, liver kidney, and nerve [51]. However, it remains unclear that how many originally delivered MSCs retain residency in the wounded tissue and maintain the appropriate terminally differentiated phenotype because large amount of transplanted population become apoptotic within the initial phase, or migrate to lungs and liver. Study on stroke and cardiac injury by Li et al. and Askari et al., respectively, suggested that transient MSCs presence appears to be sufficient to elicit a therapeutic effect [52, 53]. Taking together these findings suggests that resident MSCs also work to suppress both transient and perpetual immune surveillance systems and create an ideal healing environment by secreting factors and altering the local microenvironment [51].

Since 2002 in vitro T-lymphocyte activation and proliferation assays have been used in several studies which resulted in understanding the immunomodulatory effect of MSCs from human, murine, and baboon [54–56]. These studies demonstrated that MSCs were capable of suppressing both lymphocyte proliferation and activation in response to allogeneic antigens. Moreover, MSCs can induce development of CD8⁺regulatory T (Treg) cells than can in turn successfully suppress allogeneic lymphocyte responses [56] and prohibit differentiation of monocytes and CD34⁺ progenitors into antigen presenting dendritic cells [57]. T cells stimulated in presence of MSCs get arrested in the G1 phase as a result of cyclin D2 downregulation [58]. MSCs are also capable of inhibiting the proliferation of IL-2 or IL-15 stimulated NK cells [59, 60]. MSCs have also been shown to alter B-cell proliferation, activation, IgG secretion, differentiation, antibody production, and chemotactic behaviors [51]. Treatment with in vitro expanded allogeneic MSCs successfully resolved severe grade IV acute graft-versus host disease (GvHD) supported in vivo immunomodulatory properties of MSCs [61]. Furthermore, MSCs reduce expression of major histocompatibility complex class II (MHCII), CD40, and CD86 on Dendritic cell (DC) following maturation induction [51]. Interestingly, allogeneic MSCs which were differentiated towards a chondrogenic phenotype continued to suppress antigen specific T-cell proliferation in rheumatoid arthritis [62] and genetically engineered MSCs escaped immune rejection and induced ectopic bone formation in vivo [56]. However several other reports suggested that the immunomodulatory effects of MSCs are not universal and unconditional and that the MSCs phenotype is transient and context dependent [63].

Cytokine secretion is one of the major therapeutic characteristics of MSCs [64]. MSCs secretion is not limited to factors like TGF-β, IL-10, IL-6, cyclooxygenase-1 (COX-1), and COX-2 which are responsible for prostaglandin E2 (PEG2) secretion. MSCs partly inhibited DC differentiation through IL-6 secretion and reduced tissue inflammation by IL-10, TGF-β1, and IL-6 secretion [57, 65]. TGF-β1 secretion by MSCs suppresses T-lymphocyte proliferation and activation, initiated by IL-1β secretion from CD14⁺monocytes [66]. In fact one study suggested that only the supernatants obtained from cocultures of stromal cells and activated T cells displayed an immunosuppressive effect when added to secondary cultures of proliferating T cells [58, 67]. Taking together MSCs mediated immunosuppression is not exclusively the result of a direct inhibitory effect but involves the recruitment of other regulatory effects. Details about immune-modulation of immune response are reviewed elsewhere [68, 69].