While the above stem cell lineages may have therapeutic potential if their respective issues can be addressed, currently those limitations seriously offset their likely routine use in clinical stroke. An alternative stem cell type that is gaining recognition as a potential therapeutic are human amnion epithelial cells (hAECs). hAECs are derived from the amniotic sac, a thin avascular tissue that encloses the fetus and is attached to the placenta. The innermost layer of the amnion comprises epithelial cells that are in direct contact with the amniotic fluid. It is this amniotic epithelial layer that plays a critical role in providing the embryo with an optimal amniotic environment in which the embryo can develop.

It has been known for more than 30 years that hAECs are safe to transplant into humans [50] and they have a number of properties that make them ideal as a potential therapeutic. For example, they are anti-inflammatory [51], able to modulate host immune cell responses to injury [52], and are anti-fibrotic [53]. Indeed, amnion cells or strips of amnion membrane have long been used to reduce scarring and promote healing in burn victims [54] or following corneal surgery [55]. It is thought that the ability of hAECs to promote “scar-free” healing underlies the ability of the fetus to heal without scarring [56, 57]. hAECs are also pluripotent, including being able to differentiate down neural lineages [58]. More specifically, hAECs may express markers of glial and neuronal progenitor cells, and display multiple neuronal functions, such as synthesis and release of acetylcholine, catecholamines, and neurotrophic factors [59, 60]. Important for any future clinical therapy, and unlike ESCs, hAECs do not form tumors in vivo [50, 61], nor do they differentiate into fibroblasts as MSCs can [62], suggesting that hAECs are an inherently safer cell therapy than these other cells. Further, likely reflecting their role in maintaining maternal immune tolerance of the allograft that is the fetus in pregnancy, hAECs are immunologically inert. Primary hAECs express and release the non-polymorphic, non-classical human leukocyte antigen G (HLA-G) and lack surface expression of the polymorphic antigens HLA-A, HLA-B and HLA-C (class IA) and HLA-DR (class II) [50]. Indeed, when hAECs were administered to healthy volunteers there was no evidence of immune rejection [50]. This is in keeping with the burns and ophthalmology literature where extensive allogeneic use of amnion has occurred without rejection. Moreover, hAECs do not express telomerase and have telomere lengths more in keeping with ESCs than adult bone marrow MSCs [63]. Lastly, with approximately four million births a year (US statistics) and an average yield of 150–200 million cells per placenta [63], hAECs are abundant and relatively easy and cheap to harvest. As such, hAECs are readily available, they require no invasive procedure for harvesting, and they lack major ethical barriers to their use [32].

From the information above, it is not surprising that interest in hAECs as a source of allogeneic cells for regenerative therapies is growing [64]. In animal models of CNS disorders, accumulating evidence suggests that hAECs can exert neuroprotection and facilitate neuroregeneration [65–73]. Conversely, only one published study has tested the effect of hAECs on ischemic stroke outcome [74]. It found that direct intra-cerebral (i.c.) injection of hAECs, 24 h after middle cerebral artery occlusion (MCAO) in rats, resulted in a reduced infarct volume and improved behavioral and neurological outcomes at 16 days post-stroke. Furthermore, expression of the key marker of apoptosis, cleaved caspase-3, was reduced in the vicinity of the transplanted cells. The same research group has also reported that i.c. injection of human amnion mesenchymal cells can similarly improve stroke outcome in rats [75]. In analogous studies to those in ischemic stroke, intraventricular injection of hAECs has been reported to reduce brain edema and to improve motor deficit in a rat model of intra-cerebral hemorrhage [76]. Moreover, intra-cerebroventricular transplantation of amniotic fluid-derived stem cells at 3 days post-MCAO resulted in the attenuation of stroke-associated cognitive deficits [77]. Despite these very promising early experimental findings, i.c. injection of stem cells is not attractive as a clinical therapy for stroke patients. This is because i.c. injections would require ready access to suitable imaging facilities and surgical expertise, and could even involve significant adverse effects, such as the breakdown of the BBB and a heightened inflammatory response within the brain [78]. Furthermore, any protective effects from i.c. injection of stem cells would presumably be localized to the immediate region of brain and would not target any of the additionally detrimental stroke-induced effects occurring in the periphery. Therefore, future studies would ideally employ a less invasive and more clinically amenable delivery route of stem cells, such as intravenous (i.v.) injection.

As i.v. injection is a minimally invasive procedure, it poses a substantially lower risk of adverse clinical events when compared to i.c. transplantation and, due to the acute nature of stroke onset, an i.v. injection is ideal so that therapeutics can be administered quickly after the event. However, i.v. administration of stem cells has two initial obstacles that must be overcome: (1) the ability of the cell to pass through the extensive capillary network of the lungs; and (2) whether the cells can effectively home to stroke-affected regions of tissue in sufficient numbers to provide efficacy. Whether this may occur remains to be tested, but the relatively small diameter of hAECs approximately 15 μm) probably increases the likelihood of these cells passing through the lungs, compared with larger stem cell lineages, such as MSCs, which do not easily passage across the lungs [79]. Indeed, we have reported that only a minor percentage of i.v.-injected hAECs persist in the lungs, even if lung injury has been induced using bleomycin in mice [53]. Thus, it is conceivable that i.v.-administered hAECs may have minimal impact on lung function and that a substantial proportion of these cells can pass into the systemic circulation. In fact, Tarjiri and colleagues have reported that i.v. administration of amniotic fluid-derived stem cells at 35 days post-stroke significantly reduces infarct damage and behavioral deficits assessed at 60–63 days [80]. Hence there is considerable scope to further explore the ability of hAECs to limit injury and/or promote tissue repair and functional recovery when administered systemically following stroke.

There are numerous possible mechanisms by which hAECs could exert therapeutic effects following stroke. Firstly, hAECs could secrete neurotrophic factors that promote recovery of damaged neurons [74, 81]. Such factors could also stimulate synaptogenesis to re-innervate lost connections. Secondly, as hAECs have pluripotent properties, they could differentiate into a neuronal phenotype and replace injured or dead cells [74, 81]. Thirdly, hAECs could act as a “mobile pharmacy,” secreting necessary cytokines, growth factors, hormones and/or neurotransmitters to restore cellular function. Lastly, hAECs could potentially improve stroke outcome by modulating the immune response that contributes to brain injury [81, 82]. Included in this mechanism is the protection of neurons from immune cell-mediated apoptosis.

While it was initially proposed that stem cells exerted their reparative effects by integration and differentiation in vivo, growing evidence suggests that it is more likely that they work by modulating the host immune response to cease inflammation and coordinate resolution and repair (see Fig. 20.2). hAECs can exert immunomodulatory actions by actively suppressing T and B lymphocyte proliferation [83] reducing the expression of the potent pro-inflammatory cytokines IL-1α and IL-1β [84], and via producing inhibitors of MMPs and proteolytic enzymes associated with inflammatory reactions. In addition, although expression of HLA-G on hAECs enables their evasion of the immune system, this protein has also been shown to be anti-inflammatory by inducing apoptosis of activated CD8⁺ T lymphocytes and inhibiting CD4⁺ T lymphocyte proliferation [85]. Furthermore, hAECs transplanted to the ocular surface can create a local environment that reduces the surrounding inflammatory response [86]. This effect is thought to be due to hAECs reducing infiltration of major histocompatibility complex class II antigen-presenting cells into the inflamed cornea. Furthermore, our group has demonstrated that hAECs transplanted into a bleomycin-induced lung injury model reduces the immune response, preventing lung fibrosis and loss of function [53, 87]. These results were associated with an in vivo reduction in the pro-inflammatory cytokines, TNF, IFNγ and IL-6, and an increase in the anti-inflammatory cytokine, IL-10 [87]. As a consequence of these actions of hAECs on the immune system, there was a reduction in the infiltration of immune cells to the area of damage. In a follow-up study, we found that hAECs reduce macrophage infiltration into the bleomycin-injured lungs, reducing M1 (CD86+) macrophages but increasing M2 (CD206+) macrophages [52]. This is likely to be important to injury repair processes because, as described earlier, M1 macrophages are pro-inflammatory and are involved in the acute inflammatory response, whereas M2 macrophages are immunosuppressive and pro-reparative, coordinating the termination of inflammation, debris scavenging and angiogenesis [88]. Consistent with this, we have also shown that hAECs reduce macrophage migration and stimulate macrophage phagocytosis in vitro—features of an M2 phenotype [52].

With regard to the interactions with T cells, we postulate that hAECs could drive naïve T cells into FoxP3⁺ Tregs, consistent with a reparative response to injury, analogous to what occurs in pregnancy where the placental/fetal cells manipulate maternal immune function to prevent fetal rejection by biasing maternal immunity away from Th1 and toward Th2 activity—the so-called Th1–Th2 tilt [89]—and where the resultant Tregs drive macrophage polarity to M2 [89]. Thus, if hAECs ultimately influence macrophages to take on an M2 phenotype, and T cells to a Treg phenotype, such actions would be expected to be central to their reparative and protective effects, irrespective of where the injury is. Whether hAECs can provide neuroprotection via similar mechanisms following stroke remains to be established. Tregs certainly enter the brain following stroke where they appear to modulate tissue injury [15]. This is likely to involve the direction of non-Treg CD4⁺ T cells (T helper cells) toward a Th2-type anti-inflammatory response and away from a Th1 pro-inflammatory response [15]. Whilst there is good evidence for significant macrophage infiltration into the post-stroke brain [90], the extent of damage caused by macrophages, the identity of their phenotype/s (i.e., M1 or M2), or whether their phenotype can be manipulated to alter injury repair remains to be determined.

hAECs are thought to secrete a number of immunomodulatory factors. In fact, supernatant from hAEC culture can inhibit both innate and adaptive immune cells [83]. For example, hAECs produce alpha-fetoprotein, a protein that reduces immune cell reactivity and suppresses neuroinflammation in a mouse model of multiple sclerosis [91]. Furthermore, hAECs secrete macrophage inhibitory factor, which inhibits neutrophil and macrophage migration and natural killer cell-mediated cytolysis [83]. Fas ligand and TNF-related apoptosis-inducing ligand are both members of the TNF family that are produced by hAECs, and can regulate the immune response through apoptosis of lymphocytes [83]. Moreover, hAECs express transforming growth factor-β, which suppresses immune cell numbers through apoptosis [83]. Overall, the immunomodulatory properties of hAECs lead us to speculate that these stem cells may be able to limit the inflammatory response that contributes to infarct formation following stroke.

Stem cells communicate with each other and their environment via paracrine signaling [92]. In order to understand why and how cells migrate to their target organs, the relevant chemotactic signal(s) must be identified. Very little is so far known about the chemotaxis response involved in hAEC migration following administration. However, several studies have identified the mechanisms that attract other lineages of stem cells to injured sites following stroke. For example, there is an increase in stromal cell-derived factor-1α (SDF-1α) in brains of experimental animal models of stroke [93, 94] and a subsequent decrease in stem cell migration after the addition of an antagonist of the chemokine receptor type 4 (CXCR4) [94, 95]. SDF-1α is a growth factor produced by multiple types of mouse and human neural cells, and which functions as a chemokine that is thought to be important for neural progenitor migration during development. It is well established that the chemokine interaction between SDF-1α and CXCR4, its cognate receptor that is commonly expressed on the surface of stem cells, plays a major role in stem cell migration [94, 95]. Further research is required to clarify whether CXCR4 and/or other factors play a role in hAEC homing and signaling pathways.

Stem cell therapy was originally thought to be an opportunity to treat stroke patients in a manner that ultimately replaced dead neurons with new neurons in the infarcted region of brain. As indicated, hAECs can indeed differentiate toward a neural lineage, which may add to their potential for post-stroke therapy [32, 59, 60]. In fact, i.c. injections of hAECs in rats at 24 h after MCAO were found to migrate to ischemic areas and to then express astrocyte (glial fibrillary acidic protein) and neuronal markers (microtubule-associated protein 2 and nestin) [74]. Correlating with these observations, hAEC-treated rats showed improved behavioral and neurological outcomes, as well as reduced infarct. Thus, the authors suggested that the functional improvement following hAEC treatment may have been partially due to the newly differentiated neuron-like cells re-establishing connections with surviving host neurons. Similarly, in a hemorrhagic stroke model, hAECs transplanted into the brain were found to express neuron-specific markers and to improve motor deficits after 4 weeks [76]. As a further example, i.v. administration of amniotic fluid-derived stem cells resulted in an increased number of cells expressing microtubule-associated protein 2, and the cell proliferation marker, Ki67, in the dentate gyrus and in the subventricular zone of animals subjected to stroke, indicating increased neurogenesis [80]. Collectively, the existing evidence supports the concept that hAECs can undergo neural differentiation in vivo. Future studies must determine if these newly formed neurons are functional and able to integrate within the existing network of cells to substantially replace dead tissue.

As indicated above, secreted paracrine factors may also play a critical role in hAEC-mediated recovery after stroke. If administered as a delayed post-stroke therapy, hAECs could improve long-term stroke outcome via the release of factors that promote re-innervation, thus restoring synaptic transmitter release to stimulate plastic responses, orchestrating rescue and repair processes, and improving or preserving survival and function of existing neurons. Indeed, it has been shown that hAECs can release trophic factors such as brain-derived neurotrophic factor, neurotrophin-3, nerve growth factor [96, 97] and novel epidermal growth-like factors [98]. Whilst repair of damaged neurons following stroke has yet to be described, hAECs have shown an ability to promote recovery of injured tissue and facilitate functional plasticity in other CNS diseases. For example, transplanted hAECs produce neurotrophic substances and stimulate repair and regeneration of host neurons in a primate model of spinal cord injury [69]. In addition, hAECs transplanted into the cerebral lateral ventricle of a transgenic mouse model of Alzheimer’s disease were found to rescue damaged cholinergic neurons [99]. The authors reported that hAEC treatment increased the number of cholinergic neurons, as well as the level of acetylcholine produced by these cells, which was suggested to be largely responsible for the reversal of cognitive decline in this animal model. Therefore, hAECs may possess the ability to both repair and replace lost neuronal tissue and, together with their other anti-inflammatory characteristics, they may represent a very promising cell-based clinical therapy for neurodegenerative diseases, including stroke.