**4. Placenta-derived stem cells**

bicarbonate exchanger that exports HCO3

10 Regenerative Medicine and Tissue Engineering

/HCO3 -

cAMP and secretin stimulations [114].

deficiency, or insulin resistance [117].

Considering CFTR as a driving force for Cl-

proposed [118, 119]:

**3.2. The pathogenesis of CF liver disease**

cholangiocytes possess other ion carriers like those for Cl-

gradient of Cl-

independent Cl-


at relatively high intracellular concentrations of HCO3-

this AE (anion exchanger) activity is facilitated by the outside to inside transmembrane

stimulation. The AE activity in the liver is operated by AE2/SLC4A2 which is localized not only in the canaliculi but also in the luminal membrane of bile duct cells [105]. Experiments of RNA interference with recombinant adenovirus expressing short/small hairpin RNA have confirmed that AE2/SL4A2 is indeed the main effector of both basal and stimulated Na+

ute to intracellular pH regulation and bicarbonate secretion. Thus, CFTR had been localized at the apical side, where it plays a role in biliary excretion of bicarbonate [107, 108]. Although bicarbonate permeability through activated CFTR has been shown in several epithelia [109], its main contribution to biliary bicarbonate secretion appears to occur through a coordinated action with AE2/SL4A2 [106, 110, 111]. In addition to CFTR, cholangiocytes possess a dense population of Ca2+-activated Cl- channels. These channels are responsive to interaction of the purinergic-2 (P2) receptors with nucleotides (mainly ATP or UTP) [112, 113]. The apical fluxes of anions results in increased osmotic forces in the bile duct lumen which in the presence of AQPs contributes to water flux. AE2/SLC4A2 and CFTR colocalize with AQP1 in cholangiocyte intracellular vesicles wich coredistribute to the apical cholangiocyte membrane upon both

CF is associated with liver disease in almost 30% of all patients. In general, CF-associat‐ ed liver disease develops during the first decades of life and does not progress rapidly. The diagnostic criteria were initially established by Colombo et al. [115]. Hepatobiliary disease in CF encompass a wide variety of complications, including steatosis, focal biliary cirrhosis (FBC), multilobular biliary cirrhosis (MBC), microgallbladder, distended gallblad‐ der, cholelithiasis, intraheapatic sludge or stones, and cholangiocarcinoma [116]. The pathogenesis of steatosis (fatty liver) is not directly ascribed to the CFTR gene defect but has been attributed to malnutrition, essentially fatty acid deficiency, carnitine or choline

With regarding to the pathogenesis of FBC and MBC, various hypotheses have been

**•** *The low chloride secretion hypothesis* proposes that loss of CFTR function leads to blocked biliary ductules with thick periodic acid-Schiff positive material leading to acute and chronic periductal inflammation, bile duct proliferation and increased fibrosis in scattered portal tracts. Hepatic stellate cells (important drivers of hepatic fibrosis) become activated to produce collagen and stimulate the bile duct epithelium to produce the profibrogenic cytokine TGF-β. The progression of FBC to MBC and portal hypertension, which occurs in up to 8% of patients, may take years to decades, and should be viewed as a continuum [120].

> /HCO3 -

CF-associated hepatobiliary complications is that loss of functional CFTR protein in the

exchange, the postulated sequence of

into the bile flowing into the biliary tree. Indeed,

exchange in rat cholangiocytes [106]. Besides acid/base transporters

, Na+

, and K+

, specially upon secretin

, which greatly contrib‐


The placenta is a highly specialised organ, about 15 to 25 centimetres in diameter, that plays an important role in maintaining normal pregnancy and supporting the normal growth and development of the fetus. It is made up of a fetal and a maternal component: the fetal compo‐ nent include amnion and chorion as well as the chorionic plate, from which chorionic villi extend and make intimate contact with the uterine decidua during pregnancy; the maternal part of the placenta is the decidua basalis and it derived from endometrium.

As reported by Parolini et al. [128], different cell types can be isolated from the regions of the placenta:


In several studies hAEC, hAMSC, and hCMSC have been isolated and characterized for phenotypic and pluripotency molecular markers; moreover, has been demonstrated that these cells display differentiation potential and immunomodulatory effects [129].

hAEC express a pattern of mesenchymal markers while are negative for those of hematopoietic origin (CD90+ , CD73+ , CD105+ , CD44+ , CD29+ , CD45<sup>−</sup> , CD34<sup>−</sup> , CD14<sup>−</sup> , HLA-DR<sup>−</sup> ), and these cells are capable to differentiate in vitro into cell types of all 3 germ layers [128]. Like the amniotic epithelial fraction, the human amniotic and chorionic mesenchymal regions display the same pattern of phenotypic markers of bone marrow (BM) MSC, also displaying the expression of pluripotency markers (such as *Oct-*4) and the capability to differentiate toward different lineages including osteogenic, adipogenic, chondrogenic, and vascular/endothelial [128].

Placenta-derived stem cells seems to have a multipotent potential towards other cell types different from mesenchyme cells. hAMSC and hCMSC were shown to differentiate in vitro into a range of neuronal, oligodendrocyte and astrocyte precursors [130-132]. In addition, the use of amniochorionic membrane as a scaffold has been proposed for improving osteogenic differentiation of chorionic membrane-derived cells [133]. Alviano and colleagues reported that hAMSC display the ability to differentiate into endothelial cells in vitro [134]. Recently it has been shown that hAEC can differentiate in vitro in cells with hepatic characteristics, in particular in cells with the ability to differentiate into parenchymal hepatocytes as well as biliary cells that form duct-like three-dimensional structures when cultured on extracellular matrix [135]. hAMSC were demonstrated to differentiate into hepatocyte-like cells as judged by functional and phenotypic markers [136].

As regard the osteogenic and adipogenic differentiation of hAEC and hAMSC, discrepant results have been reported [137, 138], most likely due to the heterogeneous nature of these cell populations and due to the need to isolate the right population of progenitor cells from placental tissues. In this respect, recent efforts have been dedicated to optimizing isolation, culture, and preservation methods for placenta-derived cells; these include a study to deter‐ mine the quantity and quality of amnion cells after isolation and culture [138], while other studies aimed to define long-term expansion methods to obtain a large cell population for analysis before use in cell-based therapies.

Sources such as amnion tissue offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scalable production and long-term analysis of these cells. The establishment and characterization of human amnion-derived stem cells lines immortal‐ ized by ectopic expression of the catalytic subunit of human telomerase (hTERT) resulted in continuously growing stem cells lines that were unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differ‐ entiation potential for up to 87 population doublings [139].

Interestingly, two groups found a more reliable and unlimited non-animal source for largescale expansion of hMSC for future allogeneic clinical use: they cultured MSC with animalfree culture supplements such as human platelet lysate (PL), a suitable alternative to fetal calf serum (FCS) showing that these cells exhibit an increased proliferation potential and in vitro life span compared to cells cultured with FCS [140, 141]. On the other hand, it has been demonstrated that phenotypic shift of hAEC in culture is associated with reduced osteogenic differentiation in vitro, therefore different culturing methods may influence cell behavior [137].

In a recent comparative phenotypical study, BM- and placenta-derived mesenchymal cells has been shown that have a very similar morphology, size and cell surface phenotype for charac‐ teristics MSC markers [142]; in contrast, differences in proliferation potential have been observed between these two cell types [142]. Another study found different expressions of the chemokine receptors CCR1 and CCR3, which are only present on placenta-derived cells, while the adhesion molecules such as CD56, CD10, and CD49d have been shown to be more highly expressed on placenta-derived mesenchymal cells [143]. On the basis of numerous studies in the literature which clearly show the lack of significant differences between BM- and placentaderived mesenchymal cells types, and on the basis of the fact that placenta is readily and widely available, a good manufacturing practice-compliant (GMP) reagents and protocols has been established for isolating and expanding human placenta-derived MSC that can be directly translated to the clinical trial setup [144].

#### **4.1. Immunomodulatory features of placenta-derived stem cells**

**•** human chorionic mesenchymal stromal cells (hCMSC),

In several studies hAEC, hAMSC, and hCMSC have been isolated and characterized for phenotypic and pluripotency molecular markers; moreover, has been demonstrated that these

hAEC express a pattern of mesenchymal markers while are negative for those of hematopoietic

are capable to differentiate in vitro into cell types of all 3 germ layers [128]. Like the amniotic epithelial fraction, the human amniotic and chorionic mesenchymal regions display the same pattern of phenotypic markers of bone marrow (BM) MSC, also displaying the expression of pluripotency markers (such as *Oct-*4) and the capability to differentiate toward different lineages including osteogenic, adipogenic, chondrogenic, and vascular/endothelial [128].

Placenta-derived stem cells seems to have a multipotent potential towards other cell types different from mesenchyme cells. hAMSC and hCMSC were shown to differentiate in vitro into a range of neuronal, oligodendrocyte and astrocyte precursors [130-132]. In addition, the use of amniochorionic membrane as a scaffold has been proposed for improving osteogenic differentiation of chorionic membrane-derived cells [133]. Alviano and colleagues reported that hAMSC display the ability to differentiate into endothelial cells in vitro [134]. Recently it has been shown that hAEC can differentiate in vitro in cells with hepatic characteristics, in particular in cells with the ability to differentiate into parenchymal hepatocytes as well as biliary cells that form duct-like three-dimensional structures when cultured on extracellular matrix [135]. hAMSC were demonstrated to differentiate into hepatocyte-like cells as judged

As regard the osteogenic and adipogenic differentiation of hAEC and hAMSC, discrepant results have been reported [137, 138], most likely due to the heterogeneous nature of these cell populations and due to the need to isolate the right population of progenitor cells from placental tissues. In this respect, recent efforts have been dedicated to optimizing isolation, culture, and preservation methods for placenta-derived cells; these include a study to deter‐ mine the quantity and quality of amnion cells after isolation and culture [138], while other studies aimed to define long-term expansion methods to obtain a large cell population for

Sources such as amnion tissue offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scalable production and long-term analysis of these cells. The establishment and characterization of human amnion-derived stem cells lines immortal‐ ized by ectopic expression of the catalytic subunit of human telomerase (hTERT) resulted in continuously growing stem cells lines that were unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differ‐

, CD45<sup>−</sup>

, CD34<sup>−</sup>

, CD14<sup>−</sup>

, HLA-DR<sup>−</sup>

), and these cells

cells display differentiation potential and immunomodulatory effects [129].

, CD29+

, CD44+

**•** human chorionic trophoblastic cells (hCTC).

, CD105+

by functional and phenotypic markers [136].

analysis before use in cell-based therapies.

entiation potential for up to 87 population doublings [139].

, CD73+

12 Regenerative Medicine and Tissue Engineering

origin (CD90+

Since the placenta is fundamental for maintaining fetomaternal tolerance during pregnancy, the cells present in placental tissue may have immunomodulatory characteristics; this aspect contributes to make cells from placenta good candidates for possible use in cell therapy approaches, with the possibility of providing cells that display immunological properties that would allow their use in an all-transplantation setting.

It has been demonstrated that cells derived from placenta are negative for the expression of major histocompatibility complex (MHC) class II and for co-stimulatory molecules; all this is reflected as immune tolerance [128, 145]. Furthermore, these cells possess remarkable immu‐ nosuppressive properties and can inhibit the proliferation and function of the major immune cell populations, including dendritic cells (DCs), T cells, B cells and natural killer (NK) cells. Most of these studies have been recently summarized in up-to-date reviews [146-148]. Here, we give a brief account of the major findings concerning hAMSC.

Numerous studies showed that amniotic and chorionic membrane-derived cells can suppress the T lymphocyte proliferation induced by alloantigens, mitogens, anti-CD3 and anti-CD28 antibodies in *in vitro* and *in vivo* models [149-152]. The suppression of lympho‐ cyte population was shown to be not dependent on cell death but on decreased prolifera‐ tion and increased numbers of regulatory T cells [145]. Inhibition of T cell proliferation by placenta-derived stem cells appears to be mediated by both cell–cell interaction [153] and release of soluble factors such as indoleamine 2,3-dioxygenase (IDO), transforming growth factor β (TGF-β), and IL-10 [145, 154, 155]. The immunosuppressive activity of hAMSC on T cells seems to be not only direct but involves also DCs. Indeed, cells derived from the

mesenchymal region of human amnion impaired the differentiation of monocytes into DCs by inhibiting the response of the former to maturation signals, reducing the expression of co-stimulatory molecules and hampering the ability of monocytes to stimulate naive T cell proliferation [156]. The mechanism involved is not known, however, this inhibitory effect might be mediated via soluble factors, like IL-6, and may be dose-dependent, as it has been shown for BM-derived MSCs [157] (Figure 1).

This immune-privileged status of placenta-derived stem cells has been indicated as the cause of lack of rejection in allo- and xeno-transplantation settings. In this regard, several studies examined the fate of amniotic membrane derived stem cells grafts. Wang et al. [158] studied allogeneic GFP+ mouse intact amniotic epithelium grafts heterotopically transplanted in the eye. Kubo et al. [159] studied xenotransplanted human amniotic membrane in the eye of rats. Several preclinical studies have already reported prolonged survival of human placentaderived cells after xenogeneic transplantation into immunocompetent animals including swine [152] and bonnet monkeys [128], with no evidence of immunological rejection.

**Figure 1.** Effects of placenta-derived stem cells on immunocytes. Placenta-derived cells exert immunomodulatory ef‐ fects both on dendritic cells and T cells. Their inhibitory role is dependent on cell–cell contact and secreted soluble factors. Since most of the studies have focused on hAMSC, this cell type is represented in the scheme. iDC: immature dendritic cell; IDO: indoleamine 2, 3-dioxygenase; IL-6: interleukin-6; IL-10: interleukin-10; mDC: mature dendritic cell; TGF-β: transforming growth factor β.

#### **4.2. Clinical application of placenta-derived stem cells**

mesenchymal region of human amnion impaired the differentiation of monocytes into DCs by inhibiting the response of the former to maturation signals, reducing the expression of co-stimulatory molecules and hampering the ability of monocytes to stimulate naive T cell proliferation [156]. The mechanism involved is not known, however, this inhibitory effect might be mediated via soluble factors, like IL-6, and may be dose-dependent, as it has been

This immune-privileged status of placenta-derived stem cells has been indicated as the cause of lack of rejection in allo- and xeno-transplantation settings. In this regard, several studies examined the fate of amniotic membrane derived stem cells grafts. Wang et al. [158] studied

eye. Kubo et al. [159] studied xenotransplanted human amniotic membrane in the eye of rats. Several preclinical studies have already reported prolonged survival of human placentaderived cells after xenogeneic transplantation into immunocompetent animals including

**Figure 1.** Effects of placenta-derived stem cells on immunocytes. Placenta-derived cells exert immunomodulatory ef‐ fects both on dendritic cells and T cells. Their inhibitory role is dependent on cell–cell contact and secreted soluble factors. Since most of the studies have focused on hAMSC, this cell type is represented in the scheme. iDC: immature dendritic cell; IDO: indoleamine 2, 3-dioxygenase; IL-6: interleukin-6; IL-10: interleukin-10; mDC: mature dendritic cell;

swine [152] and bonnet monkeys [128], with no evidence of immunological rejection.

mouse intact amniotic epithelium grafts heterotopically transplanted in the

shown for BM-derived MSCs [157] (Figure 1).

14 Regenerative Medicine and Tissue Engineering

allogeneic GFP+

TGF-β: transforming growth factor β.

More than once century ago, Davis was the first to report the use of the amniotic membrane (AM) to heal skin wounds [160], prompting subsequent applications in the treatment of leg ulcers [161, 162] and burns [163], as well as for applications in ophthalmology [164]. These studies have suggested that placenta-derived stem cells may be useful for treating a range of pathologic conditions, including neurological disorders [165-167], spinal cord injury [128, 168], critical limb ischemia [169], inflammatory bowel diseases [170], and myocardial infarction [171]. Here, we will focus on the potential application of placenta-derived stem cells to lung and liver, the major organs interested by CF.
