key: cord-0913414-al9nvwet
authors: Shahrbaf, Mohammad Amin; Nouri, Masoumeh; Zarrabi, Morteza; Gramignoli, Roberto; Vosough, Massoud
title: Extraembryonic Mesenchymal Stromal/Stem Cells in Liver Diseases: A Critical Revision of Promising Advanced Therapy Medicinal Products
date: 2022-03-23
journal: Cells
DOI: 10.3390/cells11071074
sha: 6d6d5cde821e7dc518ab072a844e76ff2856e338
doc_id: 913414
cord_uid: al9nvwet

Liver disorders have been increasing globally in recent years. These diseases are associated with high morbidity and mortality rates and impose high care costs on the health system. Acute liver failure, chronic and congenital liver diseases, as well as hepatocellular carcinoma have been limitedly treated by whole organ transplantation so far. But novel treatments for liver disorders using cell-based approaches have emerged in recent years. Extra-embryonic tissues, including umbilical cord, amnion membrane, and chorion plate, contain multipotent stem cells. The pre-sent manuscript discusses potential application of extraembryonic mesenchymal stromal/stem cells, focusing on the management of liver diseases. Extra-embryonic MSC are characterized by robust and constitutive anti-inflammatory and anti-fibrotic properties, indicating as therapeutic agents for inflammatory conditions such as liver fibrosis or advanced cirrhosis, as well as chronic inflammatory settings or deranged immune responses.

There are many types of liver disease, accounting for a high burden of global diseases [1] . Liver diseases are currently a challenging issue for global public health, considering their high incidence rates, prevalence, and serious morbidities [2] . During the past several years, an increasing trend in hospitalization for chronic liver diseases has been described, particularly for end-stage cirrhosis and fatty liver disease [3] . The burden of chronic liver disease is significant in morbidity, mortality, loss of productive years, and consuming health care resources [4] . Several etiologies-including infection, alcoholism, chemical and biochemical toxins, and malignancies-are exacerbating hepatic conditions [5] . In addition, chronic liver disorders may progress in end-stage liver disease (ELD), with liver transplantation as the only effective treatment for selected patients [6] . Novel therapeutic options, including cell-based therapies and nanomedicine strategies, have been gaining attention as bridge or alternative/adjunct treatment to orthotopic liver transplantation and supporting outcomes for congenital and chronic disorders [7, 8] .

Cell-based therapies have attracted attention as promising therapeutic option, mainly due to reduced invasive clinical procedures and high regenerative potential [9] . Several reported as lacking characteristic surface proteins such as CD45 or CD34, but constitutively expressing endoglin (CD105) or membrane bound nucleotidase (CD73), in addition to glycoproteins as CD90 or intercellular adhesion molecule (CD44) when grown in in vitro conditions [14] .

Scientific literature overflows with reports and studies describing properties and characteristics of human MSCs, including their long-term proliferative capacity in 2D culture conditions, as well as critical ability to differentiate into mesoderm linage-such as bone, cartilage, and fat in permissive conditions-both in vivo and in vitro [21] .

The past decade has seen an explosion of experimental and preclinical projects directed toward better understanding of the mechanisms by which MSC act during rescue and repair of injured organs and tissues. Such a plethora of studies on MSC isolated from different tissues have been suffering from different, and frequently fluctuating, methods of isolation and expansion in vitro. Such unpredictable results were even exacerbated by inconsistent and frequently poorly effective maturation protocols performed ex vivo, aimed to generate different somatic cells, including liver cells. Over the years, several researchers and biotech companies have proposed MSCs as multipotent alternative solution to tissue-resident progenitor liver cells, bounded by genetic and epigenetic instabilities, but even more, limited by hepatic maturation level and bio-synthetic activities [22] .

Extraembryonic tissues are frequently described as perinatal tissues or as widely known placenta. Different parts of the human placenta can serve as sources of high-quality MSCs, including amniotic fluid, amnion membrane, chorion plate, the inner cytotrophoblast, outer synciothrophoblast and chorionic, yolk sac, umbilical cord tissue, and cord blood ( Figure 1 ) [23, 24] . The multipotent stem cells derived from extraembryonic tissues are largely acknowledged as valuable sources of MSCs rewarded by safety, accessibility, genome stability, non-tumorigenicity, and rescued/null ethical or religious issues for clinical application compared to the pluripotent stem cells-i.e., embryonic stem cells and induced pluripotent cells [25] . Several characteristics and functional properties have supported the use of MSCs in disparate clinical settings, including their robust potential of immunomodulatory capacities, reduced immunogenicity, differentiation into mesodermal linages, their The multipotent stem cells derived from extraembryonic tissues are largely acknowledged as valuable sources of MSCs rewarded by safety, accessibility, genome stability, non-tumorigenicity, and rescued/null ethical or religious issues for clinical application compared to the pluripotent stem cells-i.e., embryonic stem cells and induced pluripotent cells [25] . Several characteristics and functional properties have supported the use of MSCs in disparate clinical settings, including their robust potential of immunomodulatory capacities, reduced immunogenicity, differentiation into mesodermal linages, their self-renewal, and proliferative capacity (both in vivo and in vitro) [26] . Here below, we will describe in detail extraembryonic stem/stromal cells that can be isolated from human perinatal tissues, with particular attention to mesenchymal stromal cells of fetal origin. Extraembryonic MSCs role in preclinical or clinical application to reverse or correct liver disorders will be highlighted and discussed in a separate paragraph.

Amniotic fluid (AF) is an essential ingredient of amnion sac, crucial for the fetus' growth, development, and protection [27] . Such fluid is commonly harvested by amniocentesis at 15-20th week of the gestation for early diagnosis of the fetal genetic abnormalities [28] or can be collected during caesarian surgical procedure at the end of pregnancy [29] . Over the past several years, purification procedures and cytological descriptions have described the presence of cells floating in AF, primarily of fetal origin (due to skin or intestine exfoliation) or excreted within the fetal urine [30] . Amniotic fluid cells are classified into three main categories based on their morphological and biomedical activity, including the epithelioid type, AF type, and fibroblastic type cells [31] . Epithelioid types are derived from the skin and urine of the fetus and indicate a round shape and slow-growing properties. AF-type cells originated from the placenta and are associated with estrogen, human chorionic gonadotropin (hCG), and progesterone production, and fibroblast-like cells descended from mesenchymal tissue with no hormonal activity and a spindle-shaped morphology [32] .

Amniotic liquid is still considered an important source of MSC for cell-based therapy [33] . AF-MSC have been largely described and fulfilling all the criteria needed (surface markers and gene expressions) to be defined as multipotent MSC [34] [35] [36] . AF-MSCs are accessible and easily isolated/purified in a less invasive manner than other extraembryonic and somatic MSCs. However, the accessibility of these cells is associated with some concerns: first, AF-MSCs can be obtained both at mid-term through amniocentesis and at full-term delivery. However, amniocentesis procedure is considered not free from risk for the fetus and the mother; thus, such a diagnostic procedure is going to be rapidly substituted by less invasive biomolecular and serological analysis. The harvest of AF-MSC at the end of pregnancy has been shown to be attainable, but limited to caesarian section [37] . Such a practice ideally does not introduce any risk either for the newborn or the mother, but it is still poorly offered since the priority is commonly given to the baby and mother, limiting quality and quantity of AF devoted to cell purification.

Functional analysis of AF-MSC, in comparison with other sources of mesenchymal cells, proved expressions of ECM remodeling genes and adhesive factors; secretion of growth and anti-inflammatory factors have also been measured at the same level as somatic MSCs, while the expression of prostaglandins and oxytocin receptors are much higher in AF-MSC [38] . AF-MSCs proliferative capacity has also been shown at higher levels in AF-MSC, as well as their engraftment and adhesion efficiency. The same study also highlighted the reduced immunogenicity of AF-MSCs in comparison to other fetal or adult MSC [39] . Furthermore, AF-MSCs possess important ability to adapt against genotoxic stress, replicative senescence. These fetal-derived MSC have proved superior potential for DNA repair in comparison with adult bone-marrow MSCs, encouraging their application in innovative clinical setting [40] .

The clinical efficacy of AF-MSC has been mainly ascribed to their paracrine effects, such as secretion of soluble mediators (i.e., TGFβ1 and IL-10), trophic mediators, or angiogenic factors. AF-MSCs are capable to secrete soluble proteases (matrix metalloproteinases (MMP)-2, -9, and -14) responsible for ECM remodeling and fibrosis reversal [41] [42] [43] [44] . AF-MSC effects have been proved in preclinical and clinical settings, where such allogeneic cells have been offered regenerative effects for cardiovascular, renal, musculo-skeletal, gastrointestinal, hematopoietic, respiratory, neurological, and urinary diseases [45] . Furthermore, immunomodulatory and anti-oxidative effects of AF-MSCs have also been reported and described in different regenerative medicine applications. 

The human amnion or amniotic membrane (AM) is an avascular tissue, characterized in histological analysis by a thick stroma with embedded scarce MSC, while on the surface in direct contact with the fetus, epithelial cells line the surface [46] . Amnion epithelial cells (AECs) originate from epiblasts, during the second week of gestation, before gastrulation and attachment to the uterus; amnion MSC (AMSC) rise from the primitive streak of the trophectoderm, after the three germ layers have been originated [47] .

Mechano-enzymatic procedures have a proven effectiveness in isolating human AM-SCs from full-term amniotic membrane [48] . Freshly isolated or cryopreserved human AMSCs have been reported to express somehow stemness genes such as octamer binding transcription factor (Oct)-3/4, SRY (sex-determining region Y)-box (SOX)-2, Myc, Rex-1, and Nanog in addition to the angiogenic genes PECAM-1, bFGF, and VEGF. Such an expression pattern has been described decreasing during serial passages, and limited to cells at the early passage in vitro [49, 50] .

Flow cytometric analysis on human AMSC confirmed constitutive expression of surface antigens widely accepted as identity markers for MSCs (CD73, CD90, CD105) [14] . The absence of surface markers such as CD31, CD34, CD45, CD106 supports hAMSC identity and homogeneity [51] . Besides, AMSCs express human leukocyte antigen (HLA) class Ia, but lack class II (HLA-DR) [52] . These surface molecules and ectoenzymes are critical mediators to grant AMSC tolerogeneity in allogeneic settings. Human AMSC modulate activation and proliferation of host immune cells, such as T and B cells, or natural killer (NK) cells. Furthermore, extraembryonic AMSCs modulate the production of pro-inflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β, IL-5, IL-6, IL-9, IL-13, IL-17A, and IL-22 by the innate and adaptive immune cells [53] .

Amnion-derived MSCs have also been confirmed as multipotent cells, capable of differentiating into adipocytes, osteoblasts, and chondrocytes [54] . The administration of AMSCs has been described as supportive and beneficial in treating neurological, cardiovascular, and gastrointestinal disorders, but also helpful in a few cancers [55] .

Chorionic plate MSC (CP-MSC) can be isolated from the chorionic layer of the human placenta by exposing tissue to enzymatic activity. CP-MSC possess similar properties to other extraembryonic or adult MSCs, including the ability for self-renewal and mesoderm differentiation, in addition to "classical" identity proven by selective surface markers [56, 57] . The CP-MSC proliferative rate has been described superior to the afore described AMSC [58] . Furthermore, chorionic MSC present enhanced adipogenic potential [59] , described as superior to other extraembryonic MSC such as AMSC (whose osteogenic potential is instead reported preferable) [60] or umbilical-cord-derived MSC (prevalently chondrogenic) [61] . Several preclinical studies have described CP-MSC differentiation in to neuronal, pancreatic, angiogenic, and cardiomyocyte-like cells [62] .

Recent reports highlighted notable immunomodulatory properties possessed by CP-MSC and peculiar gene expressions and differentiation capacity [63] . CP-MSC have been shown particularly active in reducing T-cell proliferation and IFN-γ secretion [64] . Additionally, it has been reported that CP-MSC secrete high levels of IL-10 and TGFβ1 [65] .

The umbilical cord (UC) is a multi-layer tissue, characterized by a thick stroma with embedded blood vessels. Human UC consist of two arteries, and one vein enclosed by a gelatinous material called Wharton's Jelly [66] . Several cells can be isolated from fullterm UC, including the most described and commonly used hematopoietic stem cells floating in the umbilical cord blood [67] . Once cord blood is drained out, selective and consecutive enzymatic digestions may facilitate isolation of MSC from the Wharton's Jelly (WJ-MSC) [68] , endothelial cells from the umbilical vein (UVEC) [69] , and umbilical cord Cells 2022, 11, 1074 6 of 18 perivascular cells (UCPVC) [70] , and very small embryonic-like stem cells [71] . UC-MSC can be enzymatically isolated from Wharton's Jelly, perivascular tissue, and umbilical membrane [72, 73] . Both natural delivery and caesarean section birth have been offering quality tissues for UC-MSC manufacturing. UC-MSCs are multipotent MSCs, and as all the other somatic or extraembryonic MSCs, present characteristic morphology, plastic adherence, and certain surface markers [74, 75] . Notably, gene expression analysis in human UC-MSC resulted in highly angiogenetic and neurogenic pattern profiles compared to other adult MSCs [76] .

Human UC-MSCs have been applied in regenerative models where they enhanced innate repair capacity, induced secretion of anti-inflammatory cytokines, modulated recipient's immune recognition and rejection, and inhibited tissue apoptosis as indicated by increased Bcl-xl/Bax protein ratio and decreased cleaved caspase 3 levels [77, 78] .

The exponential growth in clinical use and commercialization of primary MSCs has been enhanced by the ethical concerns on pluripotent stem cells (such as embryonic stem cells) and rendered more attractive by past US administration prohibition in ESC study and transplant. The re-designation of MSC as stromal rather than stem cells and their refinement in paracrine properties and features did not affect their use in preclinical and clinical settings.

Considering the technical feasibility, isolation easiness, and accessible tissue, it does not surprise as extraembryonic cells and extraembryonic MSC, in particular, have been largely implemented in disparate clinical trials in previous years. The high level of heterogeneity of MSC is second only to their multiple therapeutic potency and disparate mechanisms of action. Stable and reproducible clinical outcomes require homogenic cell products, both in terms of cellular identity and potency. Nevertheless, high level of heterogeneity has been widely described as a result of different isolation methods or ex vivo cell expansion [79, 80] .

Approximately 1000 clinical trials have been registered up to 2021 [81] , using both autologous and allogeneic MSCs, for treatment of a plethora of human diseases and medical conditions. Within these 1000 MSC clinical studies, 179 studies have been conducted using (allogeneic) extraembryonic MSC (excluding umbilical cord blood cells), between 2010 and 2020. Almost 9/10 of such registered clinical trials have been conducted using UC-MSCs. Anti-inflammatory, angiogenic, and trophic effects have been called as the main impact and strength of donor cells. Multipotent differentiation capacity characteristic of primary and expanded MSC have also played an important role in some settings, particularly osteogenic and chondrogenic clinical studies. Two distinct phase 1/2a randomized controlled clinical trials have described therapeutic effects in patients with acute respiratory syndrome postinfection with COVID-19. By injecting UC-derived MSC, the clinical reports described as all anti-inflammatory and immunomodulatory effects have been significantly reduced [82] [83] [84] .

Extraembryonic MSC may represent innovative and prompt suitable cellular treatments for chronic or congenital liver disorders, if allogeneic MSC work in replacement of damaged/deficient patient's cells or enhance innate regenerative capacity (Figure 2 ).

Extraembryonic MSCs used in interventional medicine or corrective therapy would escalate and remodel current clinical strategies. Many animal studies supported the use of primary extraembryonic cells [85] . However, additional studies are required to fully characterize and profile all the characteristic and functional properties associated with primary extraembryonic MSCs purified from different parts of the perinatal tissues, and many variables can affect their characteristics and efficacy-such as donor genetic background, timing and protocol for collection samples and isolation procedures, and accurate matching between donors and recipients' needs. Preclinical and quality assessment evaluations are essential and critical for clinical settings, in support of these valuable and easily accessible source of stem cells. Extraembryonic MSCs used in interventional medicine or corrective therapy would escalate and remodel current clinical strategies. Many animal studies supported the use of primary extraembryonic cells [85] . However, additional studies are required to fully characterize and profile all the characteristic and functional properties associated with primary extraembryonic MSCs purified from different parts of the perinatal tissues, and many variables can affect their characteristics and efficacy-such as donor genetic background, timing and protocol for collection samples and isolation procedures, and accurate matching between donors and recipients' needs. Preclinical and quality assessment evaluations are essential and critical for clinical settings, in support of these valuable and easily accessible source of stem cells Over the last 20 years, a sparked enthusiasm has been generated for MSC-derived hepatocyte-like cells. Several authors reported the generation of functional hepatocytelike cells starting from mesenchymal stromal cells, lately resized but not neglected. Embryonic pluripotent stem cells have showed pluripotency, including maturation into (endoderm) hepatocyte-like cells. [86] . Somatic or extraembryonic MSC trans-differentiation into functional and secretive hepatocytes has been largely attempted, initially enthusiastically announced, but lately revised and refined since not completely corroborated by solid measurements. In the early 2000s, high impact reports described MSC-into-Hep maturation both in vitro and in vivo, persuading to clinical applications. Such studies generated results compelled by limited clinical outcome and short-term beneficial effects. Few years ago, an important report highlighted MSC-derived apoptotic bodies as main-if not sole-mediators more than intact viable cells in GvHD treatments [87] . Constitutive ex- Over the last 20 years, a sparked enthusiasm has been generated for MSC-derived hepatocyte-like cells. Several authors reported the generation of functional hepatocyte-like cells starting from mesenchymal stromal cells, lately resized but not neglected. Embryonic pluripotent stem cells have showed pluripotency, including maturation into (endoderm) hepatocyte-like cells. [86] . Somatic or extraembryonic MSC trans-differentiation into functional and secretive hepatocytes has been largely attempted, initially enthusiastically announced, but lately revised and refined since not completely corroborated by solid measurements. In the early 2000s, high impact reports described MSC-into-Hep maturation both in vitro and in vivo, persuading to clinical applications. Such studies generated results compelled by limited clinical outcome and short-term beneficial effects. Few years ago, an important report highlighted MSC-derived apoptotic bodies as main-if not solemediators more than intact viable cells in GvHD treatments [87] . Constitutive expression of adhesion molecules and chemokines, or the ability to respond to soluble chemokines, have been described as diminished or "compromised" when MSC are exposed to immunereactive or proliferative stimuli [88] .

Excitement raised during the first years of the new millennium has been reconfigured when different eminent groups confirmed inefficiency in generating mature and functional hepatocyte-like cells starting from MSC isolated by any somatic or extraembryonic source [89] [90] [91] [92] . Preclinical experiments initially interpreted as direct proof of MSC transdifferentiation into epithelial liver cells were lately corrected by fusion events occurring between donor cells and host hepatocytes. Cell fusion was shown to occur in vivo, as elegantly detailed in preclinical studies using a classical model of fumarylacetoacetate hydrolase (Fah) deficiency [93] . MSC-hepatocyte fusion has been described to be enhanced by the presence of liver injury or chronic disease or in the other disorders later tested [94] [95] [96] . Then, other groups reconsidered the generation of hepatocytes by hematopoietic cell transdifferentiation, revised in cell fusion events as the mechanism involved [97] . Several revisional studies have illustrated that the generation of hepatocyte-like cells bearing donor markers was not a consequence of MSC maturation into endoderm-like cells, rather as results of cell fusion donor cells are physio-logically prone to [39, 98, 99] . However, such melting events were not restricted to BM-MSC only, any other types of MSC, including extraembryonic-derived cells, have also been reported to fuse with hepatic parenchymal cells rather than supply new competent cells [100, 101] .

Same as the other adult MSCs, extraembryonic MSCs cells can affect different liver cells-including hepatic parenchymal cells, hepatic stellate cells, and Kupffer cells-by cell-to-cell contact, and paracrine effects [102] . MSC can support hepatocyte's proliferation and contrast parenchyma apoptosis [103] . On the contrary, donor MSCs can induce apoptosis and contrast activation or proliferation of hepatic stellate cells [104] , and liver-resident macrophages (Kupffer cells), inhibiting M1 polarization and promoting production of antiinflammatory cytokines [105] . However, despite similarities in identity and mechanisms of action among different human extraembryonic MSC isolated from different parts of placenta or amniotic fluid, such cells proved different immunomodulatory and trophic factor production potential, ascribed to different perinatal microenvironments and niches [106] .

UC-MSC can suppress T cell activation in fulminant hepatitis through chitinase 3-like protein 1 (CHI3L1) and NF-κB signaling [107] . Furthermore, the administration of UC-MSC resulted in anti-oxidant (assessed by 2 -7 -dichlorofluorescein diacetate staining) [108] , antiinflammatory, and immunomodulatory effects [109, 110] . Hepato-specific gene expressionincluding plasmatic proteins as albumin, α1 antitrypsin, or α-fetoprotein, as well as phase I mediators (i.e., cytochrome P450 subunits)-was detected in HLC derived from UC-MSC, while scarcely or inconsistently detected in BM-derived HLC [111] .

Over the past several years, clinical trials have been conducted using UC-MSC in support of hepatocyte transplantation and treatment of metabolic liver disease [112] . In clinical studies on chronic liver disorders, peripheral intravenous administration of UC-MSC was associated with decrement of aminotransferase levels and bilirubin, increase in albumin and MELD score without significant adverse effect [113] [114] [115] [116] [117] [118] [119] . Additionally, they can extend the overall survival rate in long-term follow-up [120] . Furthermore, endurance and extended donor cells have been ascribed to beneficial multiple infusion of UC-MSC rather than single injection [121] . In the context of other non-fibrotic chronic liver disorders, UC-MSC administration in models of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) resulted in hyperglycemia decrement, reversal in transaminase escalation, improvement in lipid profile (such as triglyceride, low-density lipoprotein, or total cholesterol). Improvement in NAFLD/NASH histopathology revealed reduced fat accumulation and oxidative stress in damaged hepatocytes, and reversal in microbiome diversity mediated by toll-like receptor (TLR)-4/NF-κB pathway [122] [123] [124] .

Besides umbilical cells, human primary AF-MSC have also tested as adjunct treatment for liver disorders. Ten years ago, the infusion of 10 6 rat AF-MSCs-collected between the third to the sixth passage-were administered to fulminant hepatitis model via the portal vein [125] . Such administration resulted in enhanced survival and hepatic functions (low transaminases and high serological albumin levels), reduced cell necrosis, and inflammation. In the same year, another study implanted HLCs derived from syngeneic AF-MSC into acute liver failure (ALF) model of mice, reporting remarkable hepatic engraftment and anti-inflammatory effects [126] . In another study, same amount of AF-MSC injected in a rat model of carbon tetrachloride (CCl4)-induced ALF decreased serological glutamate oxaloacetate and pyruvate transaminase, as well as reduced fibrotic areas, four weeks after cell transplantation [127] .

Recent studies observed the positive impact of A-MSC in liver diseases, including reduced inflammation, fibrosis attenuation, and pathological improvement in animal and human models [128, 129] . Human A-MSC can attenuate liver damage by paracrine anti-inflammatory cytokines [130] , as well as inhibit autophagy by Kupffer mediated by pro-inflammatory mediators [131] . Amnion-derived MSC have proved ability in preventing stellate cell activation [132] , as well as enhanced ECM remodeling mediators (i.e., MMP-2, -9, -13, and tissue inhibitor matrix metalloproteinase 1, TIMP-1) leading to fibrosis amelioration [133] . Human A-MSCs have been injected in ALF model, resulting in fibrosis amelioration, and restoring liver function (determined by histopathological and serological analysis) [134, 135] . Indeed, it has been reported as intravenous administration of human A-MSC significantly reduced hepatic fibrosis and collagen type-I deposition, prevented recruitment of CD68-positive Kupffer cells and the content of TIMP-1 in the livers in a rat model of ALF [136] . Amniotic MSC have been reported to secrete epidermal growth factor (EGF) and hepatocyte growth factors (HGF), two driving forces in liver regeneration [137, 138] .

The administration of perinatal A-MSC has been performed also in advanced fibrotic condition, such as chronic liver disorders. The transplantation of A-MSCs in a cirrhotic animal model resulted in remarkable engraftment despite deranged hepatic architecture, probably mediated by MMP-9 and MMP-13, and ameliorated liver microcirculation, reduced inflammation and oxidative stress, with consequent improvement in hepato-specific functions [139, 140] . In a recent study, the clinical effects of A-MSC have been reported in a murine model of sclerosing cholangitis, where downregulation of cytokeratin (CK)-19, MMP-9, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) have been described in association with improved pathological score [141] . In addition, A-MSC might affect hepatocellular carcinoma progression. In one study in 2020, it was demonstrated that human A-MSC could migrate to the cancer site and inhibit tumor growth. In addition, these cells express dickkopf-3 (DKK-3), dickkopf-1 (DKK-1), and insulin-like growth factor-binding protein 3 (IGFBP-3), which significantly inhibit the proliferation of cancer cells and increase the apoptosis rate of HepG2 cells. However, further studies are needed to confirm this hypothesis [142] .

In a hepatic context, also CP-MSC have anti-inflammatory, antifibrotic, and high regenerative capacity, helpful for liver disease reversal [143] . Intra-hepatic infusion of CP-MSC in CCl4-injured livers produced ECM remodeling and contained type I collagen and α-smooth muscle actin (α-SMA) expression. On the contrary, the placenta-derived cells supported expressions of albumin and MMP-9 [144] . Furthermore, intrasplenic administration of the same cells reduced the number of both apoptotic (measured by caspase 3/7 activity) and necrotic cells. However, conversely, CP-MSC increased autophagic signals and regeneration capacity, revealed by the light chain 3 II (LC 3II) increment, a marker of autophagy [145] .

Some recent shreds of evidence support the efficacy of UC-MSC derived exosomes as innovative, cell-free liver treatment factor. Intravenous or oral gavage of UC-MSC exosomes may reduce oxidative stress and apoptosis in CCl4 liver injury [146] . In addition, UC-MSC exosomes modulate CD154 expression on liver-resident T cells. The CD154 molecule has been described as an initiative factor of inflammatory response in liver ischemia/reperfusion injury [147, 148] . Furthermore, UC-MSC derived exosomes can decrease the collagen type I and III content and reduce transforming growth factor (TGF)-β1 in the liver fibrosis model [149] . In one other study in 2017, extracellular vesicles derived from human embryonic stem cell-MSCs (ES-MSC) significantly ameliorate cirrhosis in thioacetamide-induced chronic liver injury in comparison compared to bone marrow (BM)-MSC and adipose (AD)-MSC. Moreover, ES-MSC expressed anti-inflammatory cytokines and immunomodulatory activities, effective in attenuating liver fibrosis [150] .

The summary of the extra embryonic MSCs applications in liver disease is presented in Table 1 . The administration of cell-based therapies for liver disease is rapidly progressing. The growing number of registered clinical trials as well as encouraging recent market forecast reports strongly support the practical administration of quality and standardized human extraembryonic stem cells. Among several types of MSC, extraembryonic stromal cells clearly have the benefit of being accessible and non-invasive collection procedure is available. Currently, the application of these extraembryonic multipotent cells in clinical application is mainly limited by standardized manufacturing and quality assessment protocols. Cell transplantation and cellular therapies have long been the realm of university and hospital activities. However, upon validation, the cellular therapies described here could be expanded beyond liver disorders, to the other organs and wider groups of patients.

Extraembryonic stem cells possess multipotency and remarkable immunomodulatory features that may produce a paradigm shift in cell transplantation [151] . Conversely to the other current allogenic cell-based therapies, perinatal MSC do not require immunosuppression nor cause immune-reaction in the recipient. The afore described extraembryonic MSCs actively interact and crosstalk with innate and adaptive immune cells not only by cell-to-cell interactions, but also through paracrine mediators. Such mediators (collectively known as the secretome), composed by both soluble proteins and extracellular vesicles of micro-and nano-size, interact with the target cells and activate endogenous stem and progenitor cells. Both extraembryonic MSCs and their secretome have been described to suppress apoptosis, promote angiogenesis, mediate chemo-attraction, and regulate inflammatory response, stimulate the remodeling of the extracellular matrix and reduce fibrosis. A modern paradigm envisages as perinatal or extraembryonic cells do not necessarily need to mature into adult cell types (i.e., hepatocytes), but they can rescue native parenchymal cells via indirect paracrine mechanisms. 

The plasticity and functional heterogeneity of MSCs may raise potential questions in MSC-based safe and efficacious therapies in the clinical applications. An efficient and substantial proof of differentiation into functional hepatocyte (or cholangiocyte) is the core of all cell transdifferentiation studies. Extra-embryonic MSCs have a high potential for differentiation to mesodermal linage and it is proposed that they can produce HLCs, with similar morphology and physiology to the normal hepatocyte. These cells are also characterized by robust and constitutive anti-inflammatory and anti-fibrotic properties, making them suitable for inflammatory liver conditions such as hepatitis, liver fibrosis, and cirrhosis.

A major challenge in many published studies is validating progenitor/stem cell maturation into functional hepatocytes is the lack of comparison between donor-derived hepatocyte-like cells and real adult human hepatocytes. Hepatic maturation is regularly monitored by the selective upregulation and downregulation of a plethora of genes, proteins, and a few functional assays. Transcriptomic analysis may represent an easy and relatively cheap method to initially test HLCs, but such analysis needs to be performed in direct comparison with primary human hepatocytes. 

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Multilineage potential of side population cells from human amnion mesenchymal layer

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy

Characterization of amniotic stem cells

The immunomodulatory effects of mesenchymal stem cells on regulatory B cells. Front. Immunol. 2020, 11, 1843

Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy

Application of human amniotic epithelial cells in regenerative medicine: A systematic review

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies

Human chorionic plate-derived mesenchymal stem cells transplantation restores ovarian function in a chemotherapy-induced mouse model of premature ovarian failure

Different characteristics of mesenchymal stem cells isolated from different layers of full term placenta

Human chorionic-plate-derived mesenchymal stem cells and Wharton's jelly-derived mesenchymal stem cells: A comparative analysis of their potential as placenta-derived stem cells

Comparison of osteogenic differentiation capacity in mesenchymal stem cells derived from human amniotic membrane (AM), umbilical cord (UC), chorionic membrane (CM), and decidua (DC)

Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering

Chorion Mesenchymal Stem Cells Show Superior Differentiation, Immunosuppressive, and Angiogenic Potentials in Comparison With Haploidentical Maternal Placental Cells

Feasibility of placenta-derived mesenchymal stem cells as a tool for studying pregnancy-related disorders

Immunomodulatory Effects of Placenta-derived Mesenchymal Stem Cells on T Cells by Regulation of FoxP3 Expression

Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study

Stereological and histological assessment of the umbilical cord in new-born rat

Cord blood hematopoietic stem cell transplantation

Cell sheet formation enhances the therapeutic effects of human umbilical cord mesenchymal stem cells on myocardial infarction as a bioactive material

Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis

Human umbilical cord perivascular (HUCPV) cells: A source of mesenchymal progenitors

Very small embryonic-like (VSEL) stem cells: Recent knowledge

Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord

Comparison of Signaling Pathways Gene Expression in CD34(-) Umbilical Cord Blood and Bone Marrow Stem Cells

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method

Gene expression profile of mesenchymal stem cells from paired umbilical cord units: Cord is different from blood

The clinical application of mesenchymal stem cells in liver disease: The current situation and potential future

Human umbilical cord mesenchymal stem cells ameliorate acute liver failure by inhibiting apoptosis, inflammation and pyroptosis

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Onset of heterogeneity in culture-expanded bone marrow stromal cells

Advances in mesenchymal stem cell therapy for immune and inflammatory diseases: Use of cell-free products and human pluripotent stem cell-derived mesenchymal stem cells

Umbilical cord mesenchymal stem cells for COVID-19 acute respiratory distress syndrome: A double-blind, phase 1/2a, randomized controlled trial

Umbilical Cord-derived Mesenchymal Stem Cells modulate TNF and soluble TNF Receptor 2 (sTNFR2) in COVID-19 ARDS patients

Mesenchymal stem cells derived from perinatal tissues for treatment of critically ill COVID-19-induced ARDS patients: A case series

Placental stem cell correction of murine intermediate maple syrup urine disease

Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation

Bellantuono, I. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion

Failure of bone marrow cells to transdifferentiate into neural cells in vivo

Little evidence for developmental plasticity of adult hematopoietic stem cells

Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells

Preservation of hematopoietic properties in transplanted bone marrow cells in the brain

Cell fusion is the principal source of bone-marrow-derived hepatocytes

Hematopoietic mobilization in mice increases the presence of bone marrow-derived hepatocytes via in vivo cell fusion

Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Human progenitor cells with high aldehyde dehydrogenase activity efficiently engraft into damaged liver in a novel model

Cell fusion in the liver

Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners

Myelomonocytic cells are sufficient for therapeutic cell fusion in liver

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion

Mesenchymal stromal cell therapy for liver diseases

Mesenchymal stem cell-derived extracellular vesicles: Novel frontiers in regenerative medicine

What is the impact of human umbilical cord mesenchymal stem cell transplantation on clinical treatment?

Mesenchymal stem cells attenuate sepsis-induced liver injury via inhibiting M1 polarization of Kupffer cells

Designing stem cell niches for differentiation and self-renewal

Human Wharton's jelly-derived mesenchymal stem cells alleviate concanavalin A-induced fulminant hepatitis by repressing NF-κB signaling and glycolysis

Human Umbilical Cord MSC-Derived Exosomes Suppress the Development of CCl(4)-Induced Liver Injury through Antioxidant Effect

Therapeutic potential of human umbilical cord derived mesenchymal stem cells on rat model of liver fibrosis

Intravenous Umbilical Cord-derived Mesenchymal Stem Cells Transplantation Regulates Hyaluronic Acid and Interleukin-10 Secretion Producing Low-grade Liver Fibrosis in Experimental Rat

Differentiation of umbilical cord mesenchymal stem cells into hepatocytes in comparison with bone marrow mesenchymal stem cells

Human hepatocyte transplantation for liver disease: Current status and future perspectives

Effects of allogeneic mesenchymal stem cell transplantation in the treatment of liver cirrhosis caused by autoimmune diseases

The impact of recipient age on the effects of umbilical cord mesenchymal stem cells on HBV-related acute-on-chronic liver failure and liver cirrhosis

Umbilical Cord-Derived Mesenchymal Stem Cell Transplantation in Hepatitis B Virus Related Acute-on-Chronic Liver Failure Treated with Plasma Exchange and Entecavir: A 24-Month Prospective Study

Clinical therapeutic effects of human umbilical cord-derived mesenchymal stem cells transplantation in the treatment of end-stage liver disease

A study about immunomodulatory effect and efficacy and prognosis of human umbilical cord mesenchymal stem cells in patients with chronic hepatitis B-induced decompensated liver cirrhosis

Combination Treatments of Plasma Exchange and Umbilical Cord-Derived Mesenchymal Stem Cell Transplantation for Patients with Hepatitis B Virus-Related Acute-on-Chronic Liver Failure: A Clinical Trial in China

Effect of umbilical cord mesenchymal stem cell transplantation on immune function and prognosis of patients with decompensated hepatitis B cirrhosis

Mesenchymal stem cell therapy in decompensated liver cirrhosis: A long-term follow-up analysis of the randomized controlled clinical trial

Enhanced therapeutic effects of umbilical cord mesenchymal stem cells after prolonged treatment for HBV-related liver failure and liver cirrhosis

Human Umbilical Cord-Derived Mesenchymal Stem Cell Therapy Ameliorates Nonalcoholic Fatty Liver Disease in Obese Type 2 Diabetic Mice

Liraglutide in combination with human umbilical cord mesenchymal stem cell could improve liver lesions by modulating TLR4/NF-kB inflammatory pathway and oxidative stress in T2DM/NAFLD rats

Human umbilical cord-derived mesenchymal stem cell-exosomal miR-627-5p ameliorates non-alcoholic fatty liver disease by repressing FTO expression

Amniotic fluid-derived mesenchymal stem cells as a novel therapeutic approach in the treatment of fulminant hepatic failure in rats

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure

Therapeutic potential of amniotic-fluid-derived stem cells on liver fibrosis model in mice

Stem cells and liver regeneration

Amniotic mesenchymal stem cells derived hepatocyte-like cells attenuated liver fibrosis more efficiently by mixed-cell transplantation

Functional secretome analysis reveals Annexin-A1 as important paracrine factor derived from fetal mesenchymal stem cells in hepatic regeneration

Human amniotic mesenchymal stromal cells alleviate acute liver injury by inhibiting the pro-inflammatory response of liver resident macrophage through autophagy

The Application of Mesenchymal Stem Cells in the Treatment of Liver Diseases: Mechanism, Efficacy, and Safety Issues

Conditioned Medium from Human Amnion-Derived Mesenchymal Stem Cells Regulates Activation of Primary Hepatic Stellate Cells

Antifibrotic Activity of Human Placental Amnion Membrane-Derived CD34+ Mesenchymal Stem/Progenitor Cell Transplantation in Mice With Thioacetamide-Induced Liver Injury

Human Amnion Mesenchymal Stem Cells Positioned Transplantation for the Treatment of Mice Liver Damage Induced by Carbon Tetrachloride

Human Amnion-Derived Mesenchymal Stem Cell Transplantation Ameliorates Liver Fibrosis in Rats

Human amniotic mesenchymal stem cells improve ovarian function in natural aging through secreting hepatocyte growth factor and epidermal growth factor

Markers of liver regeneration-the role of growth factors and cytokines: A systematic review

Human amniotic stem cells improve hepatic microvascular dysfunction and portal hypertension in cirrhotic rats

Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse

Effects of human amnion-derived mesenchymal stem cells and conditioned medium in rats with sclerosing cholangitis

Human amniotic mesenchymal stem cells inhibit hepatocellular carcinoma in tumour-bearing mice

Isolation and characterization of chorionic mesenchymal stromal cells from human full term placenta

Anti-fibrotic effect of chorionic plate-derived mesenchymal stem cells isolated from human placenta in a rat model of CCl(4)-injured liver: Potential application to the treatment of hepatic diseases

Human placenta-derived mesenchymal stem cells promote hepatic regeneration in CCl4-injured rat liver model via increased autophagic mechanism

hucMSC Exosome-Derived GPX1 Is Required for the Recovery of Hepatic Oxidant Injury

Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Protect Liver Ischemia/Reperfusion Injury by Reducing CD154 Expression on CD4+ T Cells via CCT2

Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate rat hepatic ischemia-reperfusion injury by suppressing oxidative stress and neutrophil inflammatory response

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis

Extracellular vesicles derived from human embryonic stem cell-MSCs ameliorate cirrhosis in thioacetamide-induced chronic liver injury

Translation of amnion stem cells to the clinic

Human umbilical cord matrix stem cells efficiently rescue acute liver failure through paracrine effects rather than hepatic differentiation

Funding: This research received no external funding.

The authors declare no potential conflict of interest.