Review: Preclinical studies on placenta-derived cells and amniotic membrane: An update

Article Outline

• Abstract
• 1. Introduction
  • 1.1. Placenta-derived cells
  • 1.2. Amniotic membrane
• 2. Preclinical studies
  • 2.1. Neurological diseases
    • 2.1.1. Parkinson’s disease
    • 2.1.2. Stroke
    • 2.1.3. Spinal cord injury
  • 2.2. Pancreatic disease
  • 2.3. Muscle disease
  • 2.4. Vascular disease
  • 2.5. Pulmonary and liver fibrosis
  • 2.6. Heart disease
  • 2.7. Use of placental cells and fragments of AM for tissue engineering
  • 2.8. Enhancement of UCB-derived hematopoietic stem/progenitor cell (HSC) engraftment
• 3. Conclusions
• Financial support
• Conflict of interest
• Acknowledgments
• References
• Copyright

Abstract 

Recent years have seen considerable advances in our knowledge of the biology and properties of stem/progenitor cells isolated from placental tissues. This has encouraged researchers to address the potential effects of these cells in animal models of different diseases, resulting in increasing expectations regarding their possible utility for cell-based therapeutic applications. This rapidly evolving research field is also enriched by studies aimed at expanding the use of the whole amniotic membrane (AM), a well-known surgical material, for pathological conditions other than those tested so far and for which clinical applications already exist.

In this review, we provide an update on studies that have been performed with placenta-derived cells and fragments of the entire AM to validate their potential clinical applications in a variety of diseases, in particular those associated with degenerative processes induced by inflammatory and fibrotic mechanisms. We also offer, as far as possible, insight into the interpretation and suggested mechanisms to explain the most important outcomes achieved to date.

Keywords: Placenta, Amniotic membrane, Placenta-derived cells

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1. Introduction 

The placenta is generally recognized for important functions such as nutrition, respiration and excretion as well as maintenance of fetomaternal tolerance. The components of this organ include the fetal membranes, umbilical cord and trophoblast. In recent years, besides the use of fragments of the entire amniotic membrane (AM) as a surgical material [1], [2], much attention has been given to the different cell types that can be isolated from the placenta. Progress in understanding the biology and properties of these cells has encouraged researchers to explore their potential effects in animal models of different diseases, in the hope of future clinical applications [3], [4].

1.1. Placenta-derived cells 

Several researchers have reported the isolation and characterization, from human and animal placentas, of cell populations with the properties of stem/progenitor cells [3]. Considering the current lack of standardization regarding the isolation and cultivation of placental cells applied in different laboratories and given that, in many cases, there is no precise description of the placental regions from which the cells are isolated, it is often quite difficult to compare results reported by different groups. This is further complicated by the fact that some groups work with freshly isolated cells, while others work with cultured and expanded cells, opening the possibility that the culture conditions themselves may give rise to selection of different cell populations. This scenario is further confounded by the different developmental origins of cells isolated from human and non-human placentas. Indeed, it is important to bear in mind that, whilst the placentas of eutherian mammals share common physiological and functional features, there are remarkable differences in terms of macroscopic and microscopic structure. Generally, classification of the placental types encountered in mammals is based on two main characteristics: i) the shape of the placenta and the distribution of contact sites between the fetal membranes and endometrium (diffuse, cotyledonary, zonary or discoid); ii) the number of tissue layers intervening between the maternal and fetal blood (epitheliochorial, endotheliochorial, and hemochorial). Moreover, differences exist between animals with a similar type of placentation (e.g. human and mouse) [5], [6]. These differences should be kept in mind when selecting isolation protocols for placental cells, and especially when adapting protocols from one species to another, in order to ensure that cells are derived from the same anatomical region, thereby avoiding misinterpretations, as highlighted in some recent reports [6].

In this review, when discussing cells isolated from the human amniotic membrane, we will adopt the nomenclature of Parolini et al. [3]: human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), to indicate cells isolated from the epithelial and mesenchymal layers of the amniotic membrane, respectively, although it should also be noted that these cell populations are very heterogeneous. When describing results obtained with cells isolated from other placental regions, or cells which are defined by a specific phenotype, as well as with cells isolated from non-human placentas, we will maintain the nomenclature chosen by the authors of the studies to which we refer.

Here we will not discuss cells isolated from umbilical cord and umbilical cord blood (UCB), for which we refer readers to other reviews specifically dedicated to these cells (e.g. [7]).

Aside from their relative ease of isolation and the lack of ethical concerns for their procurement, three main characteristics of placenta-derived cells make them viable candidates for cell-based therapeutic approaches: i) their absent or low immunogenicity and immunomodulatory properties (e.g. fetal membrane-derived cells fail to induce an allogeneic T-cell response, actively suppress T-cell proliferation induced by alloantigens or by a mitogenic stimulus, block differentiation and maturation of monocytes into dendritic cells) which suggest their utility in allogeneic transplantation settings [3], [4]; ii) their multilineage differentiation capacity in vitro, even across germinal boundaries outside of their specific lineage [3], suggesting their utility in tissue regeneration approaches; and iii) their ability to successfully engraft and survive long-term in various organs and tissues, e.g. after transplantation into neonatal animals [8] and after in utero transplantation into pregnant rats [9]. Notably, human microchimerism has been detected in host organs without evidence of inflammation or rejection, indicating active tolerance of the cells [8]. Besides these important properties, it is noteworthy that placenta-derived cells can secrete a number of factors involved in various pathophysiological events, such as cytokines which have immunomodulatory and anti-inflammatory effects [10], as well as angiogenic factors associated with wound healing [10], [11], growth factors related to cell proliferation and differentiation [10], [11], [12], [13], [14], and anti-apoptotic and anti-oxidative factors [15].

1.2. Amniotic membrane 

Amniotic membrane (AM) displays anti-inflammatory properties, anti-bacterial properties and wound protection, and anti-fibroblastic and epithelialization effects (for reviews see [1], [2]). These properties, combined with the absent/low immunogenicity of AM-derived cells, has led to the use of AM as a dressing in the clinic, e.g. to promote healing of burned skin and leg ulcers, as well as to treat a continuously widening spectrum of ophthalmic disorders [1], [2]. In this review we will not include these well-known clinical applications of AM, but focus instead on recent studies where the use of fragments of the entire AM has been proposed for the treatment of other pathological conditions in preclinical settings.

This paper reviews and summarizes the majority of preclinical studies which have been performed using cells isolated from placental tissues or using fragments of the entire AM, both in allogeneic and xenogeneic settings, and offers, as far as possible, insight into the interpretation and suggested mechanisms to explain the most significant results achieved.

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2. Preclinical studies 

2.1. Neurological diseases 

Since 1996, when the group of Sakuragawa [16] suggested that hAEC may act as progenitors for neurons and glial cells, due to their expression of markers for both cell types, accumulating evidence has provoked researchers to employ direct transplantation in vivo of placental cells for the treatment of neurological disorders which affect both the brain and spinal cord.

As it was believed that differentiation of cells toward the neurogenic lineage was the first necessity for their application in vivo, several authors investigated the ability of placental cells to differentiate in vitro into neural-like and astrocytic-like cells. Indeed, placenta-derived cells, under particular culture conditions, express neuronal and glial markers [17], [18], [19]. Amniotic epithelial cells (AECs) derived from both human and non-human placentas can synthesize and release neurotransmitters and neurotrophic factors [13].

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive death of nigral dopaminergic (DA) neurons.

The majority of studies that assess the ability of placental cells to treat this disorder have been performed with hAEC in xenogeneic transplantation settings. Intriguingly, in all of these studies, despite expectations, the authors conclude that the beneficial effects conferred by hAEC are most likely related to the bioactive molecules secreted by transplanted cells, which may act by paracrine mechanisms on surrounding host tissues, rather than their ability to differentiate toward the neurogenic lineage.

Specifically, Kakishita et al. [14], [20] showed that transplantation of hAEC into an immunosuppressed rat model of PD counteracted the degeneration of nigral DA neurons [20] and afforded significant functional recovery [14], likely by means of DA and other diffusible molecules released by the transplanted cells (Table 1). A study [21] in non-immunosuppressed rats extended these results by providing evidence of a significant functional recovery, which was associated with a significant increase in DA and DOPAC (3,4-dihydroxyphenylacetic acid) levels in the striatum and of DA levels in the cerebrospinal fluid of the hAEC-treated group compared to the control group (Table 1).

Table 1. Preclinical studies for neurological diseases.

Disease	Cells	Animal Model	Manipulation in vitroa	Cell Transplantation (dose/route/timing)	Detection Timeb	Results in vivo	Suggested mechanisms		Ref
							Paracrine Mechanisms	Tissue-specific Differentiation
			culture and infection with recombinant adenovirus.	2 implants of 4 × 104 cells into denervated striatum, 2 wks after 6-OHDA treatment.	2 wks	Improvement of neurobehavioral deficit. No overgrowth of the grafted tissue.	hAEC may produce DA and other diffusible molecules with trophic and beneficial activities on DA neurons.		[14]
Parkinson’s Disease	hAEC	6-OHDA-treated rats	culture and labeling	supranigral injection of 4 × 104 cells, on the day of 6-OHDA treatment.	2 wks	Increase of nigral DA cell number. No overgrowth of the grafted tissue.			[20]
			culture	1 × 106 cells into the lateral cerebral ventricle, 4 wks after 6-OHDA treatment.	5 wks	Improvement of neurobehavioral deficit. Increase of DA and DOPAC levels.	hAEC may produce neurotrophic factors.		[21]
		MPTP-treated mice	culture and labeling	2 × 104 cells into the right striatum, 1 wk after MPTP-treatment.	4 wks	Improvement of neurobehavioral deficit. Enhanced neurogenesis in the SVZ. Preservation of DA neurons. Increase of BDNF and GDNF levels.	Transplantation of hAEC could increase BDNF and GDNF levels in striatum, with DA neuron survival and endogenous neurogenesis.		[22]
	rat AECs	gerbils with occlusion of bilateralcarotid artery	culture and labeling	implantation of 2 × 105 cells into the right dorsal hippocampus 1 wk after ischemic insult.	5 wks	Grafted cells migrated into the CA1 pyramidal layer and survived in a manner similar to CA1 pyramidal neurons.	Rat AECs could have trophic effects on damaged neurons and regenerate neuronal cells.	Rat AECs may differentiate into neuronal-like and neural stem cells.	[24]
Stroke	hAEC	rats with middle cerebral artery occlusion	culture and infection with recombinant lentivirus	8 × 105 cells into the right dorsolateral striatum, one day after stroke.	3 wks	Functional recovery, reduction of infarct area and of cell apoptosis. After transplantion, some hAEC expressed astrocytic and neuronal markers.	hAEC may release neurotrophic factors. Reduction of apoptosis of host cells.	hAEC may differentiate into astrocytic and neuronal-like cells.	[25]
	human fpMSC and mpMSC	rats with experimental stroke	2D culture and expansion in a 3D bioreactor	single and dual injection of 1 × 106 cells in the tail vein, 8h and 24h upon stroke onset.	N.D.	Functional recovery and reduction of infarct area. Improvement of astroglial reactivity. Best results with mpMSCs.	Release of soluble factors. Modulation of immunoreactions. Adjustment of astroglial reactivity.		[26]
Spinal Cord Injury	rat AECs	rats with laminectomy	culture and infection with recombinant retrovirus	transplantation, alone or with NSCs, into the injured spinal cord 7 days after SCI. Allogeneic transplantation.	5 wks	Hindlimb motor function improvement. Rat AECs promoted the survival and neural differentiation of co-transplanted NSCs, and bFGF enhanced this ability. Rat AECs supported survival of the host neurons.	Rat AECs could improve the local microenvironment of the injured spinal cord and promote the differentiation of NSCs into neuron-like cells.		[27]
	hAEC	monkeys with transection of the spinal cord.	culture and labeling	10–12 × 103 cells per mm3 into the transection cavity.	60 days	No formation of glial scar at the cut ends. hAEC graft was penetrated by the host axons.	Prevention of death in the axotomized neurons or neurotrophic effects by hAEC.		[28]
		rats with transection of the spinal cord	culture and labeling	piece of Gelfoam soaked in hAEC suspension (1 × 105 cells) into the injured site.	8 wks	Hindlimb motor function recovery. The atrophy was ameliorated and the size of injured neurons partially restored.	Release of neurotrophic factors by hAEC.		[29]

AECs: Amniotic Epithelial Cells; BDNF: Brain-Derived Neurotrophic Factor; bFGF: basic Fibroblast Growth Factor; DA: DopAmine; DOPAC: 3,4-dihydroxyphenylacetic acid; fpMSC: Chorionic Villi-derived MSCs; GDNF: Glial cell line-Derived Neurotrophic Factor; hAEC: human Amniotic Epithelial Cells; mpMSC: decidua-derived MSCs; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; N.D. Not Determined; NSCs: Neural Stem Cells; 6-OHDA: 6-hydroxydopamine; Ref: References; SVZ: SubVentricular Zone; wks: weeks.

Intra-striatal xenogeneic transplantation of hAEC also conferred benefit in a mouse model of PD [22]. These authors did not find neurons derived from hAEC in the mouse brains and suggested that transplanted cells could increase brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) levels in the striatum, promoting survival of DA neurons and endogenous neurogenesis (Table 1).

Ischemic stroke results from a transient or permanent reduction in cerebral blood flow, causing cell death within minutes. Cell therapy has been investigated as a neurorestorative treatment for this disorder [23]. Okawa and colleagues [24] showed that rat-derived AECs infused into the ischemic brains of adult gerbils could migrate into the CA1 pyramidal layer, survive and be transformed into neuronal-like cells and neural stem cells (Table 1). Subsequently [25], it was shown that hAEC transplantation into ischemic rats resulted in a significant improvement of behavioral dysfunction and reduction of infarct volume. When infected to over-express GDNF, these cells reduced the deficit more rapidly and stably than non-infected hAEC [25]. Considering that some of the transplanted cells expressed the neuronal marker MAP-2 (microtubule associated protein 2) and the neuronal progenitor marker nestin, together with the astrocyte marker glial fibrillary acidic protein (GFAP), the authors suggested that the beneficial effects exerted by hAEC could be due in part to differentiation of these cells toward the neurogenic lineage in vivo, and in part to paracrine actions of the neurotrophic factors that they secrete (Table 1).

Interestingly, Kranz and colleagues [26] have recently reported comparative results obtained after transplantation of MSCs (mesenchymal stromal/stem cells) isolated from decidua (maternal part of placenta, indicated as mpMSC) and from chorionic villi (fetal part of placenta, indicated as fpMSC) (Table 1). The authors observed that transplanted cells, mainly mpMSC, significantly increased functional recovery in treated rats with respect to control animals, with a reduced infarct ratio in mpMSC-treated subjects and an improvement in astroglial reactivity. To explain this, the authors suggested a paracrine action of the transplanted cells through modulation of peripheral and local immunoreactions, which is significant given that stroke outcome is strongly influenced by inflammatory reaction [23]. Furthermore, the authors also proposed that the transplanted cells could secrete soluble factors with anti-apoptotic, neurogenic and angiogenic effects.

Spinal cord injury (SCI) produces progressive cell death, axonal degeneration, and functional loss in multiple motor, sensory, and autonomic systems [27]. AECs have been employed to treat this disease in both xenogeneic and allogeneic settings (Table 1). When transplanted into bonnet monkey and rat models of SCI, hAEC survived and integrated in the host spinal cord, without evoking immune response or formation of scars in lesion areas [28], [29], with improvement in hind limb motor function observed in rats [29]. Although the underlying mechanisms remain to be elucidated, the transplanted cells might prevent degeneration of axotomized neurons and exert neurotrophic effects [28], [29]. In an allogeneic setting, rat AECs modified to express the basic fibroblast growth factor (bFGF) gene and co-transplanted with neural stem cells (NSCs) into a rat model of SCI, promoted the survival and neural differentiation of co-transplanted cells and supported survival of host neurons, likely by improving the local microenvironment of the injured spinal cord [27].

2.2. Pancreatic disease 

Cell-based therapy with hAEC has also been attempted in animal models of insulin-dependent diabetes mellitus (DM) (Table 2), a disease characterized by autoimmune destruction of pancreatic β-cells and lack of insulin production. Wei and colleagues [30] showed that hAEC were capable of normalizing blood glucose level in diabetic mice up to 1 month after injection. Given that transplanted human cells co-localized with human insulin secretion in the mouse tissues, the authors concluded that hAEC may differentiate into pancreatic β-cells in vivo.

Table 2. Preclinical studies for pancreatic, muscle and vascular diseases.

CLI: Critical Limb Ischemia; CP: Chorionic Plate-derived cells; DB: Decidua Basalis-derived cells; DMD: Duchenne Muscular Dystrophy; FM: Fetal Membrane; hAEC: human Amniotic Epithelial Cells; hAMSC: human Amniotic Mesenchymal Stromal Cells; MSCs: Mesenchymal Stromal/Stem Cells; N.D.: Not Determined; PLX-PAD: Placental eXpanded-Peripheral Artery Disease cells; Ref: References; Type 1 DM: Diabetes Mellitus Type 1; UC: Umbilical Cord-derived cells; VC: Villous Chorion-derived cells; wks: weeks.

Further insight into this concept came from Hou and colleagues [31] who again demonstrated the ability of hAEC to reverse hyperglycemia after transplantation into diabetic mice. Intriguingly, these positive results were obtained with hAEC which had been transplanted after induction to differentiate into functional insulin producing cells in vitro, therefore supporting the idea that differentiated hAEC may provide a source of β-cells for the treatment of insulin-dependent DM.

2.3. Muscle disease 

Recently Kawamichi et al. [32] tested the possibility of using placental cells to treat Duchenne muscular dystrophy (DMD), one of the most prevalent types of muscular dystrophy, for which an effective treatment is currently unavailable (Table 2). DMD is an X-linked recessive genetic disease caused by a deficiency in dystrophin. These authors compared the in vitro myogenic differentiation abilities of placental cell populations of both maternal and fetal origin. Only hAMSC, CP (chorionic plate-derived cells), and VC (villous chorion-derived cells) were able to express muscle-specific genes during differentiation in vitro (suggesting differentiation into myotubes). When hAMSC were transplanted into a mouse model of DMD, myofibers in the muscle tissues expressed human dystrophin and laminin. The authors suggested that this acquisition of dystrophin expression could be attributed to two different mechanisms, namely, myogenic differentiation of transplanted cells and/or cell fusion of transplanted cells with host muscle cells.

Park and colleagues [33] recently explored the in vivo myogenic and angiogenic potential of various perivascular cell populations isolated from chorionic villi, after intramuscular injection of these cells into immunodeficient dystrophic mice. The authors studied pericytes (CD146+/CD34−/CD45−/CD56−) and non-pericytes, namely, endothelial (CD146−/CD34+/CD45−/CD56−) and non-vascular (CD146−/CD34−/CD45−/CD56−) cells. Both pericytes and endothelial cells (the latter to a far lesser extent), generated differentiated dystrophin-expressing myofibers in the host, while non-vascular cells gave rise only to limited numbers of separated single myofibers. Injection of all villi-derived cells, and to a greater extent injection of pericytes, promoted local angiogenesis, likely via secretion of angiogenic factors or by recruitment of endogenous endothelial cells (Table 2).

2.4. Vascular disease 

Peripheral vascular disease (PVD) refers to diseases caused by a significant narrowing of arteries distal to the aortic arch, most often due to atherosclerosis and inflammatory processes. PVD can cause either acute or chronic ischemia, and the advanced stage is represented by critical limb ischemia (CLI). Ishikane et al. [34] investigated whether allogeneic injection of MSCs isolated from the fetal membranes of pregnant rats had therapeutic effects in a rat model of hind limb ischemia (Table 2). Three weeks after cell injection, they observed a significant improvement in blood perfusion and a higher capillary/muscle fiber ratio of ischemic muscle in transplanted groups compared with the control group. Few green fluorescent protein-positive allogeneic cells remained in the hind limb tissues, despite no evidence of endothelial differentiation or cellular fusion of transplanted cells with host cells. To explain this beneficial action, the authors suggested a paracrine mechanism whereby transplanted cells might act as a source of cytokines, which also exert angiogenic effects, and/or may have mobilized host stem/progenitor cells (e.g. endothelial progenitor cells) to the injured site to accelerate angiogenesis.

Prather et al. [35], using human placenta-derived mesenchymal-like stromal cells (named PLX-PAD for placental expanded-peripheral artery disease) expanded in a 3D bioreactor, have investigated the possibility of treating CLI in a mouse model (Table 2). Injection of PLX-PAD cells significantly improved blood flow, increased capillary density, reduced oxidative stress and endothelial damage, with a slight increase in limb function, likely through a hypothesized, though still unvalidated, paracrine secretion mechanism. Recently, data from clinical trials have shown that PLX-PAD cell therapy is safe and improves quality of life and efficacy measurements in patients with CLI (http://www.pluristem.com).

2.5. Pulmonary and liver fibrosis 

Fibrosis is defined as the overgrowth, hardening, and/or scarring of various tissues. It is attributed to excess deposition of extracellular matrix and results from chronic inflammation and an uncontrolled repair process [36]. Treatment with placenta-derived cells has been tested for pulmonary and liver fibrosis.

Lung fibrosis often represents the final stage of progression for many interstitial lung diseases, and is characterized by irreversible changes in alveolar architecture and loss of respiratory capacity.

Our own efforts have demonstrated that fetal membrane-derived cells, after both allogeneic (murine cells into mice) and xenogeneic [a mix of hAEC, hAMSC and human chorionic mesenchymal stromal cells (hCMSC) into mice] transplantation into bleomycin-challenged immunocompetent mice, by different delivery routes, significantly reduces the severity of lung fibrosis (Table 3) [37]. Concomitantly, we observed a decrease in neutrophil infiltration. Considering that these beneficial effects were observed despite a sparse presence of donor cells in host lungs, we hypothesized that these effects were not only related to engraftment and differentiation of the transplanted cells, but more to paracrine actions exerted by the soluble molecules they secreted. A different mechanism has been proposed by Moodley and colleagues [38], who suggest that the beneficial effects of hAEC on lung fibrosis could result from adoption of a lung phenotype in vivo by transplanted hAEC and by an overall anti-inflammatory effect of these cells that modifies the response of the mouse lung to injury (Table 3). Specifically, they demonstrated that primary hAEC express lung-associated markers in vitro, and when cultured in a particular differentiation medium, produce and release surfactant proteins and display lamellar bodies typical of type II pneumocytes. When transplanted into bleomycin-instilled immunocompromised mice it was found that transplanted hAEC became positive for surfactant protein expression in the lungs of injured mice, suggesting their differentiation into type II pneumocytes in vivo. hAEC injection was associated with reduced levels of pro-inflammatory cytokines [interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α] and of a pro-fibrogenic cytokine [transforming growth factor (TGF)-β] in the lungs. Moreover, hAEC transplantation reduced fibrosis and collagen deposition and induced a collagen-degrading environment (altered proteases levels) in the injured lungs.

Table 3. Preclinical studies for lung and liver diseases.

α-SMA: alpha-Smooth Muscle Actin; ALT: Alanine AminoTransferase; AM: Amniotic Membrane; FM: Fetal Membrane; hAEC: human Amniotic Epithelial Cells; hAMSC: human Amniotic Mesecnhymal Stromal Cells; hCMSC; human Chorionic Mesenchymal Stromal Cells; IL-: InterLeukin-; MMP-2: Matrix MetalloProteinase-2; Ref: References; TGF-β: Transforming growth factor-beta; TNF-α: Tumor Necrosis Factor-alpha; wks: weeks.

Since Sakuragawa and colleagues [39] first demonstrated that albumin- and α-fetoprotein-producing hAEC are promising transgene carriers for allogeneic transplantation into liver, other work has encouraged the use of placental cells for restoring functionality of hepatic tissues. For example, human AM-derived cells have been shown to engraft in the liver [8] and differentiate toward the hepatogenic lineage in vitro and in vivo [3], [17].

Liver fibrosis is a common outcome of a variety of chronic liver diseases following different insults, such as viral infection, alcoholism, chemical toxicity or metabolic and biliary disorders. Manuelpillai and colleagues [40] have recently investigated the effects of hAEC transplantation in a mouse model of CCl₄-induced liver fibrosis (Table 3). These authors found hAEC in host liver, as well as in the spleen, lung and heart of some animals. After hAEC transplantation, a significant decrease was observed for hepatocyte apoptosis, hepatic inflammation and fibrosis, with a reduction in the hepatic content of pro-inflammatory cytokines (TGF-β, IL-6 and TNF-α), an increased expression of the anti-inflammatory cytokine IL-10 and collagenases, as well as a reduction of the number of activated collagen producing hepatic stellate cells.

We have recently demonstrated the value of fragments of the entire AM for treatment of biliary fibrosis in a rat model of bile duct ligation (BDL) (Table 3) [41], providing evidence that fragments of human AM, when applied as a patch onto the rat liver surface, significantly reduce the severity and slow the progression of liver fibrosis. The exact mechanism whereby human AM patches exert these beneficial effects on BDL-induced liver fibrosis remains to be defined. Since no human cells were detected in AM-treated rat livers, it is conceivable that the effects exerted by AM patch application might not be due to a replacement mechanism whereby AM-derived cells present in the host liver differentiated toward the hepatogenic lineage, but rather to the release of soluble factors by cells of the AM patch with paracrine effects on host tissue.

2.6. Heart disease 

Although the ability of placenta-derived cells to differentiate toward cardiomyocytes is still debated [3], [4], [42], some groups have tested the ability of these cells to treat cardiac diseases.

Fujimoto et al. [43] investigated the potential of syngeneic transplantation of rat AM-derived cells (ADCs) into a rat model of myocardial infarct (Table 4). Transplanted cells were found in the host cardiac tissues at all time points studied, and remarkably, reduction of myocardial scarring and prevention of myocardial thinning was observed. The authors suggested a two-fold explanation for these results: i) differentiation of transplanted cells into cardiomyocytes and/or into vascular endothelial and smooth muscles cells; ii) stimulation of angiogenesis and prevention of residential cardiomyocyte death by transplanted cells. A similar double-faceted explanation was offered by Ventura and colleagues [44] to explain the ameliorating effects observed after transplantation of human fetal membrane-derived MSCs, which had been pre-treated with a cardiogenic/vasculogenic agent, into infarcted rat hearts (Table 4).

Table 4. Preclinical studies for other pathological conditions.

ADCs: Amnion Derived Cells; AM: Amniotic Membrane; F.M. : Fetal Membrane; FM-MSCs: Fetal Membrane-derived MSCs; hAMSC: human Amniotic Mesenchymal Stromal Cells; MSCs: Mesenchymal Stromal/Stem Cells; N.D. Not Determined; PLX-1: Placental-derived and expanded mesenchymal-like stromal cells; Ref: References.

Our recent investigation into the utility of human AM patching to repair cardiac ischemia in a rat model [42] demonstrated that fragments of the entire AM could significantly reduce postischemic cardiac dysfunction (Table 4). Indeed, when a fragment of human AM was applied to the left ventricle of rats that had undergone ischemia, these rats showed higher preservation of cardiac dimensions and improved cardiac contractile function in terms of higher left ventricular ejection fraction, fractional shortening, and wall thickening, over a two month follow-up period. As we did not detect human cells in the host tissues, we believe that the beneficial effects were due not to differentiation of the transplanted cells, but more likely to release of mediators by the AM-cells that promoted protection and regeneration of host tissues.

2.7. Use of placental cells and fragments of AM for tissue engineering 

Given that placenta-derived MSCs, like those derived from other sources, are able to differentiate in vitro toward the chondrogenic lineage [3], the use of these cells has been investigated for repair/regeneration of cartilage defects in vivo, which is a frequent defect with limited self-healing. Zhang and colleagues [45] inserted human chorionic villi-derived MSCs, which had been pre-embedded in a collagen sponge and cultured for two weeks in chondrogenic medium, into the articular osteochondral defect of nude rats (Table 4). Six weeks after surgery, the original defect was covered with reparative tissue and the bottom part of this tissue showed hyaline cartilage appearance. However, the edge of the reparative tissue showed hypertrophic cartilage formation, suggesting that while placental cells could be used to regenerate/repair damaged cartilage, it is necessary to set up appropriate transplantation conditions to avoid side effects. Recently, Wei and colleagues [46] also demonstrated that hAMSC, when implanted with collagen scaffolds into the cartilage defects of nude rats, underwent characteristic morphological changes concurrently with deposition of collagen type II, suggesting their differentiation into chondrocytes in vivo (Table 4).

Recently Ismail et al. [47] investigated the possible utility of fragments of the entire human AM as a bile duct substitute to repair three types of bile duct damage in a dog model, in the presence or absence of a vascularized support, with beneficial outcomes (Table 4).

2.8. Enhancement of UCB-derived hematopoietic stem/progenitor cell (HSC) engraftment 

In patients with damaged or defective bone marrow (BM), BM transplantation or intravenous infusion of BM-derived HSC has become a standard approach. However, as it is not always possible to find a suitable, matched donor, alternative strategies are being developed. One of these is the use of UCB-derived HSC. Although this approach is effective for pediatric patients, in adults it results in delayed engraftment due to limited cell doses that can be recovered from UCB. Therefore, strategies to enhance engraftment of UCB-derived HSC are needed. Recently, Hiwase and colleagues [48] demonstrated that co-transplantation of human placental MSCs into NOD/SCID mice enhanced engraftment of CD34+ cells isolated from human UCB, both when CD34+ cells were obtained from a single UCB and when these cells were isolated from two UCBs (Table 4). By using a dual labeling strategy of cell tracking, these authors demonstrated that the injected cells homed to the BM. Similar experiments in the same animal model, have been reported by Prather et al. [49] who used a higher dose of human placenta-derived mesenchymal-like stromal cells (named PLX-I by the authors) expanded in a 3D bioreactor (Table 4).

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3. Conclusions 

It is clear that the range of potential clinical applications of placenta-derived cells and of fragments of the entire AM is continuously widening and evolving.

Although, initially, differentiation of placenta-derived cells to specific lineages was considered the first necessity for their therapeutic application in vivo, with the idea that these cells should replace defective cells and regenerate damaged tissues, it is now emerging that the beneficial effects may more likely be due to secretion of bioactive molecules that could act on other cells and on the microenvironment which they occupy, promoting endogenous tissue repair or eliciting other beneficial effects (anti-inflammatory, anti-scarring, angiogenic, etc.) through paracrine actions. Nevertheless, the two mechanisms (tissue-specific differentiation versus paracrine actions) are not mutually exclusive and both can account for the observed improvements. Although many of the observations from preclinical models support the hypothesis of a paracrine mechanism of action of placental cells and fragments of the entire AM, in most cases this remains a hypothesis arising very often from purely descriptive results, with no molecular dissection of mechanisms, components or target cells of these mechanisms, and with many vital pieces of information still lacking. Therefore, elucidation of these mechanisms is paramount to future clinical applications.

It is conceivable that in the future, cell-free treatments based on the use of culture medium conditioned by placenta-derived cells, rather than treatment based on the use of cells per se, will represent a novel therapeutic strategy which could potentially complement or replace cell transplantation.

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Financial support 

This study was supported by a grant from Fondazione Cariplo and from MIUR.

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Conflict of interest 

The authors declare that they have no conflicts of interest of any kind with respect to any of the data/information reported in this review.

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Acknowledgments 

The authors express their gratitude to Dr. Marco Evangelista, who has provided invaluable help in the revision of this review.

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References 

1. Parolini O, Soncini M, Evangelista M, Schmidt D. Amniotic membrane and amniotic fluid-derived cells: potential tools for regenerative medicine?. Regen Med. 2009;4:275–291
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