Adipose-Derived Stem Cells and Their Potential to Differentiate into the Epithelial Lineage

Abstract

Adipose-derived stem cells (ASCs) possess a multilineage differentiation potential, can be used from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared with other sources. It is easily accessible in large quantities with a minimal invasive harvesting procedure, and the isolation of ASCs yields a high amount of stem cells, which is essential for stem cell-based therapies and tissue engineering. Differentiation of ASCs into cell types of mesodermal origin has been shown in a variety of studies. The plasticity of ASCs toward cells of the mesodermal lineage has been shown by their differentiation into chondrocytes, osteoblasts, adipocytes, and myocytes. Their potential to differentiate into lineages with nonmesodermal origin is even more exciting: ASCs are also able to differentiate into cells of ecto- and endodermal origin. Various in vitro and in vivo studies documented the induced differentiation into neural cells, hepatocytes, pancreatic islet cells, endothelial cells, and epithelial cells. Epithelial cells can embryologically arise from each of the 3 germ layers. This article summarizes and discusses the current knowledge of the potential of ASCs to differentiate into the epithelial lineage. The differentiation of ASCs into different types of epithelial cells, including hepatocytes, pancreatic cells, and endothelial cells, is highlighted together with a view on current clinical trials and future options.

Introduction

R esearch on stem cell sources for tissue engineering or for therapeutical interventions for tissue regeneration support has accelerated during the past 2 decades. The scientific community is currently searching for optimal and safe stem cells for widespread medical treatments. Adult stem cells seem to be the preferred candidates for therapeutical approaches, especially because adult stem cells hold no ethical concerns and can be isolated in appropriate amounts from several sources. Mesenchymal stromal (stem) cells (MSCs) are a stem cell population found in nearly all adult tissues (eg, bone marrow, adipose tissue, synovium, dermis, periosteum, and deciduous teeth), in peripheral blood, menstrual blood, and in solid organs (eg, liver, spleen, and lung) [1 –3]. MSCs possess a multilineage differentiation potential, are useable from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues. At present, there is a strong amount of data indicating that MSCs represent an independent population of stem cells with self-renewal properties and a multipotent differentiation profile [4 –6]. In general, MSCs are isolated by their capacity to adhere to culture-dish plastic. The cells can be expanded in culture while maintaining their multipotency during standard cell culture, and are phenotypically characterized by a specific panel of markers (minimal criteria: expression of CD73, CD90, and CD105, and lack of CD11b or CD14, CD 19 or CD79α, CD45, and HLA-DR expression [7]).

In 2001 and 2002, the initial work of Zuk and co-workers first characterized the multipotent character of MSCs from adipose tissue [the so-called adipose-derived mesenchymal stromal (stem) cells (ASCs)] [5,8]. The cells can be easily separated from subcutaneous adipose depots by different washing, digestion, and centrifugation steps, followed by the outgrowth of the plastic adherent fraction [8]. Adipose depots are ubiquitously accessible in large quantities with a minimal invasive procedure (liposuction aspiration), and contain high amounts of ASCs, which is an essential prerequisite for stem cell-based therapies. It has been described that stem and progenitor cells in the primary isolates [the so-called stroma-vascular fraction (SVF)] usually amount to up to 3%, and this is 2,500-fold more than the frequency of MSCs in bone marrow (up to 0.002%) [9]. Others described that a bone marrow transplant contains ∼6×10⁶ nucleated cells per mL [10], of which a maximum of 0.001%–0.01% are stem cells [11]. By comparison, 0.5–2.0×10⁶ SVF cells can be isolated from per gram of adipose tissue from subcutaneous liposuction aspirates [8,10,12 –14]. The associated percentages of stem cell yield range from 1% to 10% [13,15,16], likely being determined by donor and tissue harvesting site.

ASCs: Tissue Localization and Heterogeneity in Cultures

The localization of ASCs within adipose tissue is not totally clarified. Several studies have tried to identify the localization of the stem cell population within intact adipose tissue. This is a complicated endeavor because no single marker specifically and unequivocally identifies undifferentiated ASCs. Histological studies using immunohistochemical and immunofluorescence techniques suggest that ASCs reside within adipose tissue in a perivascular location [17 –22], where ASCs coexist with pericytes and endothelial cells. It has also been speculated that ASCs (and MSCs in general) are a subset of pericytes or vascular precursor (stem) cells at various stages of differentiation located in the wall surrounding the vasculature [23]. Whereas many studies provided evidence and it seems likely that ASCs reside in a perivascular niche in a CD34⁺/CD90⁺/CD31⁻/CD45⁻/CD146⁻ phenotype [18,24,25], the definite identification of the ASC population(s) in situ or in the SVF has currently not been achieved. This is indeed based on the fact that the term ASC is related to the plastic adherent and cultured population that dramatically changes the phenotype very early during cell culture [24,26 –28]. It seems obvious that these changes in the phenotype of the cells—decrease in the expression of some antigens and increase of others—are induced by adherence to plastic, culture conditions, and time in culture.

ASC preparations per se are heterogeneous cell cultures comprising a subset of stem cells (or different subsets of stem and progenitor cells) and more differentiated cells (endothelial cells, smooth muscle cells, and pericytes). Heterogeneity of MSC isolations and cultures has been shown and discussed in many publications [29 –31]. Nevertheless, cell culture selects for a more homogeneous cell population, which is enriching for cells expressing a stromal immunophenotype [15]. The expression of some characteristic surface markers by cultured ASCs was consistently found in nearly all studies (eg, CD10, CD13, CD29, CD44, CD49e, CD73, CD90, CD105, and CD166), whereas other markers are consistently not found to be expressed (eg, CD11a, CD11b, CD14, CD31, CD45, and HLA-DR) [32]. Nonetheless, studies also differ in their characterization of some of the markers studied: the expression of some antigens is described very contradictory in these studies (summarized in ref. [32]). Some reports describe CD34, CD54, CD106, or CD146 to be expressed on cultured ASCs, whereas others do not find the expression of these antigens. These differing results are due to differences in the isolation or culture method, or caused by the investigation of different passages of cultured ASCs. Although CD34, for example, is reckoned to be a hematopoietic stem cell-associated marker, it is expressed by early passages of ASCs' subsets and subsequently lost in later passages [15,25]. Its downregulation may be related to the physiological process of commitment and/or differentiation from an immature status into more specific progenitors [33]. The extent and time course of the loss of CD34 expression strongly depends on culture conditions, such as plating density and culture medium [33]. It has also been described that CD34 expression on quiescent stem cells might be upregulated in response to proliferation signals and, as such, may be a marker of activated and self-renewing stem cells [34]. In addition, it has been shown that the SVF consists of a CD34⁺ and CD34⁻ subset [33]. CD34⁺ cells are more proliferative and have a higher ability to form colonies, whereas CD34⁻ cells have a greater ability to differentiate into adipogenic and osteogenic lineages [33].

In summary, ASC cultures seem to differ between different laboratories or different isolations, and this may be due to differences in the isolation or the culture procedure. It has been reported that liposuction side, liposuction procedure, age, or body mass index play an important role in the cell yield, growth, and frequency of stem cells [13,35 –38]. All these variables may affect the composition of the isolated initial cell culture, but it is extremely difficult, if not impossible, to standardize these variables. On the other hand, the methods and quality of ASC isolations from different laboratories per se vary tremendously, resulting in a different composition of the initial cell culture. Heterogeneity of the initial cell cultures can be reduced by a washing procedure early in the beginning of the cell culture [39], indicating that several subsets require different time points to adhere to the cell culture plastic. Another effort to reduce the heterogeneity or to isolate specific subsets of ASCs was done by using flow cytometric sorting or immunomagnetic separation, either by positive (eg, anti-CD271) or by negative selection (eg, anti-CD45 or anti-CD31) [39 –42]. The usage of such techniques for the reduction of heterogeneity is slightly beneficial, but leads to a very small cell yield. By using immunomagnetic beads, Rada and co-workers demonstrated that the SVF is composed of several subpopulations, which express different levels of ASC markers and exhibit varying osteogenic and chondrogenic differentiation potential [41]. It has also been shown that the CD105⁺ population from cultured ASC exhibit a much stronger chondrogenic potential than CD105⁻ cells [42].

Finally, the culture procedure of isolated ASCs differs between the laboratories; at the present time there is no unique and standardized culture protocol for ASC culture. There are many variables that may impair the outgrowth of different cells from the initial cell culture or influence cultured cells in their undifferentiated state: the initial plating density, coating of culture dishes, composition of cell culture basal media and their glucose concentration, cell culture supplements (bovine serum, human serum, platelet lysate, or growth factors), addition of antibiotics, method of subculturing, and method of storage (cryopreservation). Limited information has recently become available about which medium optimally expands ASCs by maintaining the undifferentiated stem cell character in vitro [43 –45]. Many laboratories use Dulbecco's modified Eagle's medium (DMEM) as a basal medium to culture ASCs, but there are different DMEM commercially available. A further description of the exact medium used in these studies is often not indicated. We use DMEM with an approximately physiological glucose content (100 mg/dL). Others use a basal medium with a higher glucose content because ASCs show a much better proliferation rate in this medium. Nevertheless, a physiological glucose content is one variable that should be considered to be near to the in vivo situation. Further, most of the investigators use fetal calf or bovine serum as a culture medium supplement. Related to a possible use of ASCs in human therapeutical approaches, there are many concerns in practicability of fetal calf or bovine serum (infectious complications, host immune reactions) [46]. Also, human serum may be a source of pathogen contamination or immunoreactivity, and shows batch-to-batch variability. Using fully defined cell culture medium is an urgent need to produce ASCs for clinical applications. The gold standard for culturing ASCs will be a medium absolutely free of animal serum or factors, with well-known ingredients, that is yet not available in a satisfying quality.

In summary, modifications in the isolation and/or culture conditions might select for the expansion of subpopulations and have a huge impact on the differentiation potential of the cells cultured, although the primary cells could be phenotypically identical [31]. Therefore, standardization of the isolation and culture procedure is highly desirable for the good reproducibility of results from different laboratories and studies. In addition, good manufacturing practices-qualified isolation and culture protocols are needed for the use of ASCs in clinical trials [47,48].

ASCs Are Able to Differentiate into Cells Derived from All Germ Layers!

The potential of ASCs to differentiate into cell types of mesodermal origin has been shown in a variety of studies: ASCs can be induced to differentiate into chondrocytes, osteoblasts, adipocytes, and myocytes (cardiomyocytes, and smooth muscle and skeletal muscle cells) [5,6,8,9,49 –54]. This seems to be obvious because ASCs themselves are of mesodermal origin. As there are several reviews that summarize the differentiation of ASCs into the mesodermal lineage [32,55 –59], this aspect is not repeated here. In general, the induction of ASCs' differentiation in vitro is mainly achieved by culture in selective media with lineage-specific induction factors. The transcriptional and molecular events triggering the mesodermal lineage-specific differentiation of stem cells are well known, and are described or reviewed elsewhere [58,60 –64]. Interestingly, ASCs have also been shown to be angiogenic and act as hematopoietic supporting cells [65 –67]. These supporting characteristics of ASCs are mainly due to the secretion of antiapoptotic, angiogenic, and hematopoietic factors (cytokines and growth factors), such as the macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, vascular endothelial growth factor (VEGF), hepatocyte growth factor, and transforming growth factor-β [68 –70].

Even more exciting is their potential to differentiate into lineages with nonmesodermal origin. ASCs are also able to differentiate into cells of ecto- and endodermal origin. A number of studies have documented the induced in vitro differentiation into neural cells, hepatocytes, pancreatic islet cells, endothelial cells, and epithelial cells [65,68,71 –79]. It has, therefore, been speculated that ASCs may be pluripotent rather than multipotent [55].

Epithelial Differentiation

Although epithelial cells can arise from each of the 3 germ layers (and are embryologically not only of ecto- and endodermal origin), ASCs' differentiation potential into the epithelial lineage is the main focus of this review. Epithelial cells are commonly characterized by a cytokeratin-based cytoskeleton, strong intercellular connections, and a pronounced cell polarity. In contrast, mesenchymal cells have no such distinct intercellular connections and are, therefore, not polarized as such, and express a vimentin-based cytoskeleton [80]. Undifferentiated ASCs do not express cytokeratin filament proteins or other epithelial-specific proteins such as zonula occludens protein-1, occluding, and E-cadherin. However, both epithelial and mesenchymal cells show some degree of reciprocal cell plasticity, a phenomenon well known in tumor progression, fibrosis, and morphogenesis, as well as in tissue repair [epithelial–mesenchymal transition (EMT) or, in reverse, mesenchymal–epithelial transition (MET)] [80].

The multiorgan engraftment of transplanted ASCs has been shown in combination with epithelial lineage differentiation [81]. Fang and co-workers examined the in vivo characteristics and behavior of human ASCs transplanted in sublethally irradiated nonobese mice with diabetes or severe combined immunodeficiency. By using immunofluorescence stainings (pan-cytokeratin) and in situ hybridization (Y chromosome), differentiation of transplanted human ASCs into epithelial cells of the gastrointestinal tract, liver, and bronchi, and endothelial cells has been demonstrated. The results further suggest that no cell fusion of transplanted ASCs with epithelial or endothelial cells was responsible for their specific tissue cell-like differentiation. These results highlight the epithelial differentiation potential of ASCs in principle. Nevertheless, in vivo studies with more specific tissue or organ injury models are needed to verify this capacity.

Epithelial Differentiation Potential of ASCs In Vitro and In Vivo

Beginning in 2004 with the verification of ASCs' differentiation capacity into endothelial cells [67,76], morphologically a specialized epithelium, several studies have verified the differentiation potential into the epithelial lineage in the following years (Table 1).

Table 1.

The Potential of Adipose-Derived Stem Cells to Differentiate into the Epithelial Lineage

ASC differentiation into	Authors	Model	Verification
Epithelial cells (in general)	Brzoska et al. [77]	In vitro	Cytokeratin 18 ↑, vimentin ↓
	Baer et al. [78]	In vitro	Cytokeratin 18 ↑, CD90 ↓, α-smooth muscle actin ↓
	Long et al. [79]	In vitro	E-Cadherin ↑, cytokeratin 8 ↑
Dermal/epidermal epithelial cells	Ebrahimian [86]	In vitro/in vivo	Cytokeratin 5 ↑, cytokeratin 14 ↑
	Nie et al. [87]	In vivo	Pan cytokeratin ↑
Renal epithelial cells	Li et al. [83]	In vivo	Pan cytokeratin ↑
Retinal epithelial cells	Vossmerbaeumer et al. [85]	In vitro	Cytokeratin 8 and 18 ↑, Bestrophin ↑, retinal pigment epithelium protein 65 ↑
Vocal fold epithelial cells	Long et al. [84]	In vitro	Functional evaluation of elasticity, microstructure, and phonation
Endothelial cells	Planat-Benard et al. [76]	In vitro/in vivo	CD31 ↑, von Willebrand factor ↑/incorporation into vessels, neovascularization
	Miranville et al. [67]	In vitro/in vivo	CD31 ↑, von Willebrand factor ↑/Increased blood flow and capillary density, incorporation in the vasculature
	Cao et al. [90]	In vitro/in vivo	CD31 ↑, CD34 ↑, eNOS ↑, VE-cadherin ↑, acLDL uptake/neoangiogenesis
	Moon et al. [91]	In vitro/in vivo	Van Willebrand factor ↑, formation of vessel-like structures/increased vascular density, increased perfusion index.
	Uysal et al. [93]	In vivo	von Willebrand factor ↑
	Zhang et al. [92]	In vitro	CD31 ↑, von Willebrand factor ↑, eNOS ↑, VE-cadherin ↑, acLDL- and lectin-uptake
	Nie et al. [87]	In vivo	CD31 ↑
Endocrine pancreatic cells	Timper et al. [73]	In vitro	Insulin ↑, glucagon ↑, somatostatin ↑, pancreatic transcription factors (Isl-1, Ipf-1, Ngn3) ↑, ABCG2 ↓
	Lee et al. [94]	In vitro	C-peptide ↑, Sox17 ↑, IPF-1 ↑
	Okura et al. [95]	In vitro	Insulin ↑, C-peptide ↑, further pancreatic genes ↑
	Chandra et al. [96]	In vitro/in vivo	Insulin ↑, glucagon ↑, somatostatin ↑/restoration of normoglycemia
	Lin et al. [97]	In vitro/in vivo	Insulin ↑, glucagon ↑, NeuroD ↑/lowered blood glucose, higher glucose tolerance
	Kajiyama et al. [98]	In vivo	Insulin ↑, C-peptide ↑, restoration of pancreatic function
Hepatocytes	Seo et al. [71]	In vitro/in vivo	Albumin ↑, α-fetoprotein ↑, LDL uptake, urea production/albumin ↑
	Talens-Visconti et al. [105]	In vitro	CD90 ↓, albumin ↑, cytochromes ↑, liver specific transcription factors ↑
	Talens-Visconti et al. [106]	In vitro	Liver-specific genes ↑, change in morphology
	Banas et al. [72]	In vitro/in vivo	Cytokeratin 18 ↑, transthyretin ↑, α-fetoprotein↑(and other hepatic markers), LDL uptake (and other functional markers)/GPT ↓, improved ammonia concentration
	Sgodda et al. [101]	In vitro/in vivo	Cytokeratin 18 ↑, CD26 ↑, albumin ↑, cytochrome P450↑(and other hepatic markers)/CD26 ↑, engraftment
	Banas et al. [99]	In vivo	Improved liver function, markers of liver injury ↓
	Banas et al. [100]	In vitro/in vivo	Albumin ↑, tryptophan-dioxygenase ↑, acLDL uptake/markers of liver injury ↓
	Aurich et al. [102]	In vitro/in vivo	Urea formation, glycogen synthesis, carbamoylphosphate synthetase ↑, albumin ↑, cytochrome P450 ↑/albumin ↑, HepPar1 ↑
	Ruiz et al. [103]	In vitro/in vivo	Cytokeratin 19 ↑, albumin ↑, further liver specific genes ↑, glycogen storage/Engraftment
	Okura et al. [104]	In vitro/in vivo	Albumin ↑, acLDL uptake, urea production cytochrome P450 activity, glycogen storage/improvement of serum albumin and total bilirubin levels

↑, expression upregulated; ↓, expression downregulated; ABCG2, ATP-binding cassette, subfamily G, member 2; acLDL, acetylated low-density lipoprotein; eNOS, endothelial nitric oxide synthase; GPT, glutamic-pyruvic transaminase; IPF-1, insulin promotor factor-1.

To differentiate ASCs into renal tubular epithelial cells, the epithelial differentiation potential of ASCs in general have begun to be explored. These studies have clearly verified that ASCs can enter the epithelial lineage when treated with retinoids [77] or conditioned medium (CM) from renal tubular epithelial cells [78,82]. ASCs treated with retinoic acid or CM reduced the expression of CD90 and the mesenchymal marker vimentin and started the de novo expression of epithelial cytokeratin 18. Cytokeratin filament proteins are one of the first epithelial-specific structural proteins to be synthesized in the epithelial differentiation program. On the other hand, vimentin is an intermediate filament protein used as a molecular marker for mesenchymal cells. In addition, preliminary results from these recent studies have shown that it is also possible to induce the expression of tight junctional protein zona occludens protein 1 in cultured ASCs (own unpublished results). Nonetheless, it is currently not possible to induce in vitro other epithelial-specific markers such as E-cadherin, claudins, and occludin. In vivo differentiation of ASCs toward epithelial cells has been shown in a renal ischemia-reperfusion model [83]. Human ASCs were transplanted in kidney-impaired mice to observe the homing and differentiation of ASCs, and to elucidate the related mechanisms. Using immunohistochemistry and polymerase chain reaction analysis of human β-actin, Li and co-workers convincingly demonstrated the homing into the injured kidney and the differentiation of culture-expanded ASCs into the epithelial lineage.

In addition, 2 in vitro studies by Long and co-workers also showed that ASCs are able to differentiate into an epithelial phenotype [79,84]. The authors cultured ASCs in a fibrin scaffold (in Transwell inserts on the medium-air-interface) with growth factors, creating a 3-dimensional construct with a bilayered cell structure. In this tissue-engineered construct, ASCs treated with epidermal growth factor (EGF) near the surface of the fibrin gel differentiate into an epithelial phenotype expressing E-cadherin and cytokeratin 8, whereas deeper cells within the fibrin gel expressed vimentin, indicating their mesenchymal phenotype.

ASCs' epithelial capacity was further highlighted by a study inducing more specialized epithelial cells in vitro. A culture of ASCs with vasoactive intestinal protein or with CM from retinal pigment epithelial (RPE) cells induced differentiation into RPE cells [85]. The authors verified this differentiation by the expression of epithelial cytokeratins and typical RPE markers (bestrophin, RPE 65). In addition, RPE-differentiated ASCs displayed an enhanced stimulation of pigment synthesis by melanocyte-stimulating hormone [85].

Ebrahimian and co-workers demonstrated that ASCs participate in dermal wound healing via an epithelial differentiation process [86]. ASCs were induced to express epidermal markers cytokeratin 5 and 14 in vitro. Green fluorescent protein (GFP)-positive ASCs incorporated in dermal and epidermal tissue in vivo and promoted reepithelialization of skin wounds in mice. This effect on skin regeneration relied not only on ASCs ability to produce specific growth factors, but also on their differentiation toward a keratinocyte phenotype and into endothelial cells. Further, Nie and co-workers demonstrated in an excisional wound-healing model in rats that locally administered ASCs accelerate wound-healing through epithelial and endothelial differentiation and vasculogenesis [87]. GFP-labeled ASCs were applied to wounds to determine whether ASCs could differentiate along multiple lineages of tissue regeneration in a specific microenvironment. Immunofluorescence stainings indicated that GFP-positive ASCs costained pan-cytokeratin and CD31, respectively, indicating differentiation into epithelial and endothelial lineages. The positive effect of ASCs on wound-healing has also been shown in earlier studies, but none of these studies have shown convincingly that administered ASCs differentiate into the epithelial lineage. Nevertheless, cutaneous wounds treated with ASCs demonstrated improved keratinocyte migration, vascularization, and healing [88,89].

The epithelial potential of ASCs may open a wide range of therapeutic options, not only to regenerate wounded skin and other epithelial cell layers but also to facilitate tubular epithelial cell regeneration after renal injury.

Endothelial Differentiation

Because the vascular endothelium is a specialized type of epithelium, it should also be discussed here. The capacity of ASCs to differentiate into endothelial cells has been shown. Planat-Benard and co-workers demonstrated that cultured human ASCs differentiate into endothelial cells in vitro, incorporate into vessels, and promote both postischemic neovascularization in nude mice and vessel-like structure formation in a Matrigel plug [76]. Others demonstrated that a subset of ASCs (CD34⁺/CD31⁻ cells) is able to differentiate into endothelial cells if cultured in endothelial growth medium supplemented with insulin-like growth factor and VEGF [67]. In this study, endothelial differentiation was shown by the upregulation of the von Willebrand factor and CD31 expression. Nevertheless, the low-level expression of endothelial cell-associated markers (CD31, VE-cadherin, VEGF receptor 2, and von Willebrand factor) has been described on undifferentiated ASCs [15]. Miranville and co-workers also demonstrated that an intravenous injection of ASCs in mouse ischemic hind limb is associated with an increase in the blood flow and the capillary density and an incorporation of the cells in the leg vasculature. In contrast to the study by Miranville, a study by Cao and co-workers isolated a CD34⁻/CD31⁻ subset from the SVF and showed that this subset also expresses endothelial markers if cultured in the presence of VEGF [90]. The authors demonstrated in vivo that these cells are able to contribute to neoangiogenesis in a hind limb ischemia model. ASCs' differentiation potential into endothelial cells was further validated in several in vitro and in vivo approaches [91 –93]. The potential of ASCs to differentiate into endothelial cells is clinically useful to improve neovascularization of ischemic tissues and organs.

Pancreatic Cell Differentiation

Another example of cells of the epithelial lineage is pancreatic endocrine cells. Timper and co-workers induced ASCs to differentiate into a pancreatic endocrine phenotype using the differentiation factors activin-A, exendin-4, hepatocyte growth factor, and pentagastrin [73]. They observed downregulation of the stem cell marker ABCG2 and upregulation of the pancreatic transcription factors Isl-1, Ipf-1, Pax-6, and Ngn3, together with induction of the endocrine islet hormones insulin, glucagon, and somatostatin. This differentiation into pancreatic cells has been verified in subsequent studies [94 –96]. Okura and co-workers showed the in vitro differentiation by the induction of 16 specific markers, including E-cadherin, the transcription factor pancreatic duodenal homeobox 1 (Pdx1), insulin, glucagon, and somatostatin, during their stage-specific differentiation program [95]. The authors also showed cells coexpressing insulin and C-peptide by immunohistochemistry and the production of insulin in response to glucose by an immunoassay.

It has further been shown that ASCs that were previously transduced with Pdx1, upregulated insulin, glucagon, and NeuroD secreted an increasing amount of insulin in response to an increasing concentration of glucose [97]. Transplantation of these cells under the renal capsule of pancreatic damaged rats resulted in lowered blood glucose and higher glucose tolerance. Kajiyama and co-workers verified these results in a mouse model [98]. They used a mouse model of pancreatic damage to evaluate the pancreatic repair potential of ASCs. Transplanted ASCs were previously transduced with Pdx1. The results suggest that Pdx1-transduced ASCs firmly engrafted in the pancreas, acquired a functional insulin-producing beta cell phenotype, and partially restored pancreatic function in vivo [98]. In the future, application of ASCs may open a promising cell therapeutic option in the treatment of diabetes.

Differentiation into Hepatocytes

The differentiation potential into hepatocytes, 1 of the 2 epithelial cell types in the liver, has been shown in response to hepatocyte growth factor, oncostatin M, and dimethyl sulfoxid [71]. After incubation, ASCs expressed albumin and α-fetoprotein, produce urea, and take up low-density lipoprotein, indicating differentiation into hepatocytes. ASCs were also able to integrate into the liver of mice in a model of liver injury and expressed albumin in vivo [71]. A number of in vitro and in vivo studies verified the hepatic differentiation potential of ASCs [72,99 –106]. The in vitro upregulation of epithelial- and hepatocyte-specific markers, including cytokeratin 18 and 19, α-fetoprotein, and cytochrome P450, have been shown in response to hepatogenic induction by cell culture media supplements. Yamamoto and co-workers provided a comprehensive analysis of the process underlying the differentiation of ASCs into hepatocytes [107]. Analysis of gene ontology groups indicated that 1,639 genes were up- or downregulated, and many of these genes belong to gene ontology categories relevant to hepatic function. The epithelial markers E-cadherin and α-catenin were upregulated during differentiation of ASCs into hepatocytes, whereas the mesenchymal markers N-cadherin and vimentin were downregulated. Further, the morphology changed to an epithelial-like morphology and the expression of 2 EMT regulators, Twist and Snail, was downregulated.

Transplantation of ASCs into an in vivo model of liver injury showed that ASCs improved liver functions [72,99,100]. This was verified by the measurement of ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations and their return to a near-normal level [99]. A publication from the same group confirmed these results by a following study [100]. In vivo differentiation of transplanted ASCs into hepatocytes has been shown by immunostainings for human albumin [72]. More insights into the in vivo differentiation of transplanted human ASCs were given by the following studies. A study by Ruiz and co-workers demonstrated that human ASCs engrafted robustly into the mouse liver-expressed human albumin and human-specific α1-anti-trypsin, 2 markers of hepatic differentiation [103]. Aurich and co-workers presented immunostainings for specific markers for hepatocytes (albumin and HepPar1) after transplantation of either undifferentiated or predifferentiated human ASCs into mice [102]. The costaining of albumin-positive human cells with Connexin 32 was shown to demonstrate functional cell–cell communications between human and mouse cells in the liver, indicating integration and differentiation of human ASCs. Further, engraftment was improved using ASCs predifferentiated to hepatocytes in vitro compared with undifferentiated ASCs.

The hepatocytic potential of ASCs could be used to improve liver regeneration by cell-based therapies or to generate fully differentiated hepatocytes for ex vivo tissue engineering.

Molecular Mechanisms of Epithelial Differentiation

The molecular mechanisms underlying ASCs differentiation into the epithelial lineage are not fully understood. Only a few in vitro studies have been done to explore the signaling pathways, second messengers, and transcription factors related to ASCs epithelial differentiation. The induction of ASC differentiation is mostly carried out by the addition of growth factors and/or other lineage-specific factors to the culture medium. For example, the combination of VEGF and basic fibroblast growth factor has been described to induce endothelial differentiation of ASCs [90]. Inhibition of phosphatidylinositol-3-kinase by a specific inhibitor blocked the differentiation into endothelial cells in vitro, indicating that this pathway is essential for ASCs endothelial differentiation. An inhibitor of the mitogen-activated protein kinase (MAPK) did not affect this differentiation [90,92]. On the other hand, CM from renal tubular epithelial cells induced epithelial differentiation of ASCs via MAPK activation [78]. Nevertheless, it has not been tested whether this epithelial differentiation is blocked by an MAPK-inhibitor.

Moreover, Yamamoto and co-workers analyzed the process underlying the differentiation of ASCs into hepatocytes induced by a mixture of growth factors [107] by gene ontology analysis. They showed that 1,639 genes were significantly up- or downregulated (at least 10-fold alteration). The data indicated that many hepatocyte-related genes were upregulated and showed that MET occurred during ASCs differentiation into hepatocytes. MET as well as its reverse process, EMTs, play pivotal roles during organ development, and described to be re-engaged in adults during tissue regeneration, organ fibrosis, and cancer progression [80,108,109]. The expression levels of Twist and Snail, known to be transcriptional activators of EMT, were downregulated during ASCs hepatocytic differentiation process in vitro [107]. Another study investigated the differentiation of ASCs into hepatocytes induced by lineage-specific growth factors using microarray analysis [110]. The results also confirmed the involvement of molecular pathways of METs [110].

Moreover, direct delivery of specific key transcription factors has been used to differentiate (or reprogram) ASCs into the epithelial lineage. Functional insulin-producing beta cells were differentiated from Pdx1-transduced ASCs [97,98].

Another promising approach to investigate the molecular mechanisms underlying epithelial differentiation of ASCs could be conducted by using microRNAs (miR). It has been described that stable overexpression of miR-200a switched mesenchymal cells to an epithelial state, accompanied by a significant reduction of stem-like cell features [111]. Members of the miR family are known to be strong inducers of epithelial differentiation by post-transcriptional repression of the EMT activators zinc-finger enhancer binding transcription factor-1 and -2 (ZEB-1 and -2) [112]. ZEB-1 and -2 are, besides the transcription factors snail and slug, the main repressors of E-cadherin expression. Therefore, direct inhibition of these transcription factors may a promising effort to induce ASCs epithelial differentiation and to study the involved mechanisms.

The potential of bone marrow MSCs (BM-MSCs) to differentiate toward the epithelial lineage has also been shown [113 –117]. Although MSCs are able to differentiate into epithelial cells, only limited information about the underlying mechanism controlling this process is available. In the most studies this differentiation was only verified by morphological changes and the expression of 1 or 2 markers.

Wang and co-workers demonstrated the induction of cytokeratin 18, occluding, and a transmembrane regulator in a co-culture condition of rat BM-MSCs and airway epithelial cells. In this condition, induction of the epithelial differentiation was accompanied by a downregulation of Wnt/β-catenin [118]. Therefore, blocking Wnt/β-catenin signaling promoted MSCs to differentiate toward epithelial cells [118]. Medium with Wnt3a or lithium chloride inhibited the epithelial differentiation, whereas the loss of β-catenin induced by the Wnt-antagonist Dickkopf-1 enhanced it. Ke and co-workers explored the role of Wnt-signaling on BM-MSCs differentiation into hepatocytes [119]. Addition of Wnt-1 inhibited the differentiation into hepatocytes, whereas blocking Wnt-1 signaling by Dickkopf-1 induced BM-MSCs to express albumin [119]. Therefore, these results highlight the role of Wnt signaling inhibition on BM-MSCs hepatocytic differentiation.

Clinical Trials

Apart from all the proven basic scientific evidence in in vitro and in vivo studies that have been accumulated during the last few years, we are at the beginning of a new era of stem cell therapy. ASCs are probably one of the most powerful stem cells. Both preclinical studies and clinical trials using either SVF cells or ASCs have been initiated for autologous or allogenic therapeutical trials (recently reviewed in ref. [57]).

The first clinical application described, a case report, was an autologous transplantation of ASCs in a 7-year-old girl suffering from widespread calvarial defects after a severe head injury [120]. Using autologous ASCs in a fibrin glue, bone formation and complete calvarial continuity 3 months after the reconstruction was demonstrated. At present, ASCs are clinically applied in the treatment of diabetes, liver regeneration, fistula, fecal incontinence, spinal cord injury, lipodystrophy, ischemia, and vascular diseases (www.clinicaltrials.gov). Immunomodulatory therapy using ASCs for graft-versus-host disease and other autoimmune diseases is also currently recruiting participants. The basis for an immunomodulary therapy are the described immunosuppressive properties of ASC [121]. ASCs do not possess HLA class II antigens and are able to modulate the T-cell activity [121].

Further, cultured predifferentiated ASCs have recently been used to engineer large synthetic bone grafts and to study the vascularization process in vivo. Only limited results from these clinical studies have been published recently [122,123]. Impressive results were shown by the first large, multicenter, randomized phase II clinical trial using ASCs to treat fistulas in Crohn's disease [122]. Fistula healing was observed in 71% of patients who received ASCs in addition to fibrin glue compared with 16% of patients who received fibrin glue alone. In another publication, these investigators described the histological and electron microscopical findings from 5 biopsies taken up to 2 years after ASC implantation to provide further support for the safety of ASC therapy [123]. The results clearly showed fistula healing at the implanted area, although the ASC-mediated mechanisms responsible for this improvement could not be defined. Transplanted ASCs were not labeled for safety reasons. Thus, identification of transplanted ASCs was impossible. Nevertheless, electron microscopies from biopsy specimens (taken 19 and 24 months after transplantation) showed normal tissue with highly vascularized areas, and areas of stratified epithelium and normal ultrastructural features. Whether differentiation of ASCs into epithelial cells is involved in the process of fistula healing is unsolved. It is also not clear whether the epithelial differentiation of ASCs into pancreatic islet cells or into hepatocytes plays a role in the recent clinical trials to treat diabetes [124,125] or severe hepatic dysfunction [126].

The mechanisms of action remain unclear. In general, there are different possible models of action. One is the infiltration with the subsequent integration and differentiation of ASCs into the injured tissue. Another is a paracrine effect on surviving, resident cells induced by the secretion of cytokines, growth factors, and other cellular factors. Evidence has also indicated that the regenerative effects are mediated by angiogenic, antiapoptotic, and growth factors secreted by ASCs [68,69,127 –130]. ASCs migrated into an injured or diseased organ or tissue may secrete these factors that stimulate recovery in a paracrine manner. Further, both mechanisms (paracrine stimulation and differentiation along a desired lineage) may exist in parallel. Another possible mechanism is that ASCs stimulate the endogeneous stem cells of the host in a paracrine manner and, therefore, modulate the stem cell niche [59].

Future Directions

Active clinical applications using ASCs as therapeutical agents have only recently begun, but much remains to be done to verify the efficacy, the mechanisms of action, and safety of ASCs in treating humans. Additional information is also required concerning the therapeutic efficacy of transplanted cells by using pretreated (or predifferentiated) cells. Further, ASCs' culture and scale-up procedures need to be standardized. As all these fields are addressed, new applications will be developed leading to novel therapeutic options. One promising approach, for example, is the administration of ASCs for the treatment of acute renal (tubular epithelial) injury. In vitro expanded ASCs may also be a therapeutic option for the repair or regeneration of other epithelial tissues, for example, epithelial cells of the respiratory tract, the bladder, or dermal tissue, through direct differentiation into epithelial cells, through regeneration-promoting indirect effects, or through tissue engineered grafts.

Finally, ideal MSCs for use in therapeutical approaches need to be isolated with minimal harm to the patient, must be available in high cell numbers, proliferate in culture, and differentiate into a broad spectrum of lineages. ASCs fulfill these requirements.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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