Cardiovascular Therapeutic Applications of the Human Amnion: Membrane,Cells,and Beyond

Abstract

Background

The therapeutic potential of the human amnion has been known since the early twentieth century. Subsequent study has revealed the further therapeutic potential of all elements of the amnion—membrane, cells, fluid—in the treatment of cardiac disease.

Materials and Methods

A systematic review was performed utilizing PubMed/MEDLINE and Embase with search terms including “amniotic fluid,” “cardiovascular disease,” “cardiac disease,” “amnion,” “amniotic membrane,” and “heart.” Results were reviewed by each author to ensure inclusion of all relevant articles. Animal studies were included for evaluation of existing preclinical models, and the few available clinical studies of amniotic products were included.

Results

Preclinical studies addressing organ function, assessment, and enhancement of cardiac performance in response to injury, and regenerative potential are included, as are the few clinical studies utilizing amniotic products for the treatment of cardiac disease. Therapeutic approaches include reduction of inflammation, immunomodulation, and the promotion of myocardial regeneration via cellular therapy to target the most common mechanisms underlying myocardial injury.

Conclusions

The components of the human amnion have anti-inflammatory, immunomodulatory, and pro-differentiation abilities which lend the ability to attenuate myocardial ischemia-reperfusion injury, temper cardiac fibrosis, and promote activation of progenitor cells to induce regeneration. Preclinical studies have focused heavily on cellular therapy, but clinical experience has yielded little success. The acellular components of the amnion have fueled more recent investigation and represent a new source of enthusiasm for clinical translation of amniotic products in the treatment of cardiac disease.

Keywords

amniotic fluid cardiovascular disease cardiac disease amnion amniotic membrane heart

Introduction

The human amnion is the innermost of the fetal membranes and consists of 3 main components: amniotic membrane (AM), individual amniotic cells and their cellular products, and amniotic fluid (AF), which suspends the human fetus. Documented use of amniotic products dates to the early twentieth century when AF was first implemented in clinical practice to reduce postoperative peritonitis, and later to treat a variety of orthopedic conditions.^1–3 Since that time, the use of amniotic products for clinical therapeutic applications has naturally expanded.

To date, scant information exists regarding the influence of amniotic products in cardiac disease. Direct application of the AM has been utilized to prevent pericardial adhesions, and cell therapy with placenta-derived cells (human amniotic epithelial cells [hAECs]) and human-derived mesenchymal stem/stromal cells has been explored for promotion of cardiac regeneration.^4–6 More recently, there is a growing interest in the acellular components of the amnion and their ability to attenuate myocardial ischemia-reperfusion injury, temper cardiac fibrosis, and promote activation of progenitor cells to induce regeneration.^7–13 Herein, we review the therapeutic role and influence of amniotic products, and, in particular, AF as they relate to cardiac disease.

Materials and Methods

This systematic review was performed utilizing PubMed/MEDLINE and Embase with search terms including “amniotic fluid,” “cardiovascular disease,” “cardiac disease,” “amnion,” “amniotic membrane,” and “heart.” Results were reviewed by each author to ensure inclusion of all relevant articles. Animal studies were included for the evaluation of existing preclinical models.

The Human Amnion—Membrane and Cells

Histologically, the AM consists of 3 components: (1) an epithelial cuboidal cell layer which lines the amniotic cavity and comes into direct contact with the AF, (2) a thick basement membrane which contains multiple types of collagen lending exceptional strength and flexibility, and (3) an extensive avascular stroma which contacts the chorion and receives nutrients obtained via the chorionic villi and their communication between trophoblasts/syncytiotrophoblasts and maternal endometrial tissue (Figure 1).¹⁴

The AM's extensive basement membrane has garnered attention as a potential scaffold and bolster material for broad tissue repair applications. Its mechanical properties as well as its demonstrated anti-inflammatory and scar-reducing activity have led to its use as a dressing and tissue graft in skin/wound and burn care, abdominal closure material in the treatment of gastroschisis, tissue graft in the treatment of spinal cord injury, corneal grafts in ophthalmology, autograft in vaginoplasty, and even tendinous and osseous repair in orthopedics.¹⁵

More recently, however, it is the cellular component of the amnion that has most excited the field of regenerative medicine. The cells of the human amnion are derived from 2 areas—the placenta and the AF—and, hence, are classified as such. The placental cells consist of the innermost epithelial cuboidal cells of the AM (hAECs), the mesenchymal stromal cells within the AM (human amniotic mesenchymal stromal cells [hAMSCs]) deep to the epithelial cell layer, the human chorionic mesenchymal stromal cells, and the human chorionic trophoblastic cells. The AF cells consist of the mesenchymal stem cells (MSC) (human AF-derived MSCs [hAFMSCs]) located within the AF.¹⁶

It is worth noting that the terminology and abbreviations associated with the various cell types are often inconsistently referenced within the literature.¹⁷ In general, their individual classification and isolation depends largely on their anatomical location and the identification of surface markers as indicators of their potency.^16,18,19 In accordance with consensus guidelines developed at the International Workshop on Placental-Derived Stem Cells, we will henceforth adhere to the aforementioned classification and abbreviations based on anatomical location and potency of the cells.²⁰

The cells of the human amnion have primarily been investigated for their regenerative potential and stem cell-like properties, given their early cellular “age.” Multiple studies have subsequently confirmed not only their multipotency^19,21–23 but also the ability to acquire induced pluripotency with exposure to cellular reprogramming factors.⁴ In addition, these cells possess low immunogenicity, nontumorigenicity, high genetic stability, and fewer ethical and regulatory issues related to their use compared to embryonic stem cells (ESCs), making them ideal from a tissue engineering perspective.^14,18,21,22 This has led to considerable attention and investigation for regenerative therapeutic applications in nearly every organ system. Amniotic-derived cells—whether they be placental or AF derived—have demonstrated potential for regeneration via both peripheral and myocardial neovascularization and angiogenesis, cerebrovascular ischemic insult modulation, pancreatic cell differentiation, and skin growth, to name a few.^24–28 Indeed, this has been one of the most fervently investigated areas of modern tissue engineering research.

Despite the attention and excitement paid to the cells of the amnion, the acellular component has not been as extensively studied but has elicited substantial interest in the last decade. Extracellular vesicles (EVs) and the acellular AF itself represent the newest foci of amniotic product research in the treatment of cardiac disease. The bulk of the liquid component of AF is largely derived from maternal blood, but it does have some contribution from the AM cells. The AF fills the amniotic cavity and surrounds the growing embryo to provide germ-free protection and nourishment throughout gestation with a total volume of ∼800 mL to 1L at the time of birth. The AF contains both soluble and insoluble components forming what has been referred to as the “ideal, germ-free bath, cushion and liquor for the fetus.”²⁹ This ubiquitous fluid has previously been utilized for its antiadhesive, pro-angiogenic, anti-inflammatory, and nutritive properties in the clinical treatment of human wounds, musculoskeletal injuries, COVID-19, and ocular graft-versus-host disease but has otherwise been minimally used in the treatment of cardiac disease.^30,31

Human Amnion in Heart Disease

Amniotic Membrane

The AM contains an impressive basement membrane with a robust supply of supportive connective tissue. This extensive array of tissue lends mechanical integrity and strength, which make it naturally appealing for use as a structural support adjunct. Furthermore, with the known pro-angiogenic, anti-inflammatory, and pluripotent qualities of its cells, AM may represent a material with great therapeutic potential for the treatment of heart disease, and, in particular, the ventricular remodeling and fibrosis seen after ischemia.^14,21,32

A prior systematic review thoroughly evaluated the use of AM in the theater of cardiac disease and nicely summarized the current state of results regarding its use.³² The authors concluded that while the hypothesized effects of increased angiogenesis, reduction in infarct size, and the paracrine influence of AM have been substantiated in animal studies, there is a discrepancy in the degree of cardiomyocyte differentiation that AM produces and disagreement as to the functional impact it yields. In models of acute myocardial ischemia, AM appears to have a greater potential to induce cardiomyocyte differentiation than in models of chronic myocardial infarction, but in both, the resultant change in myocardial function does not appear to be linked to the degree of cardiomyocyte differentiation.^33–35 Consequently, therapeutic applications seek to capitalize on the AM's provision of mechanical strength and local paracrine factors to: physically support the myocardium, augment myocardial hypoxia tolerance,^36,37 reduce structural changes associated with myocardial remodeling,^38–41 improve angiogenesis in infarcted tissue zones,^42–46 reduce inflammation and adhesion formation,^6,47–49 and, ideally, promote cardiomyocyte differentiation.^33,50,51 Modern therapies utilizing the AM appear predominantly in 2 forms: (1) topical patch/scaffold and (2) injectable hydrogel.

Topical patch and scaffold applications often explore the use of AM as a vehicle for localized cellular delivery. Multilayered decellularized human fetal heart tissue + acellular AM has been used to create a “cellular sandwich” to deliver neonatal ventricular cardiomyocytes^46,52 2 weeks after permanent right coronary artery ligation with resultant increase in angiogenesis and a reduction in fibrosis with persistent survival of implanted cardiac cells.⁴⁶ Similarly, an epicardial acellular AM patch improved cardiac function and reverse remodeling in a rat model of heart failure 7 days after LAD ligation.⁵² In an alternative approach, which seeks to deliver AM beyond the epicardium, AM was packaged as an injectable hydrogel to promote deeper uptake in injured myocardium. Here, a scaffold of decellularized AM was seeded with MSCs and injected into the peri-infarct zone. One study using this approach noted improved homing and survival of MSCs in the infarcted myocardium with a greater percentage of viable, surviving MSCs following combined injection with the AM versus MSCs alone.⁵¹ Such coadministration of decellularized AM paired with a cellular source highlights the synergistic potential for AM to improve cell viability via paracrine influence to promote cell survival. Hydrogel injection represents an emerging therapeutic option for myocardial infarction and is subject to ongoing exploration. Table 1 refers to some of these studies of acellular amniotic products for treatment of heart disease.

Table 1.

Preclinical Cardiac Applications of Acellular Products of the Human Amnion.^a

Amniotic component	Model	Findings
Acellular hAM⁵¹	Acute MI without reperfusion: Permanent LAD ligation Injectable hAM hydrogel paired with mesenchymal stem cells (single dose immediately after ligation)	- In-vitro: hAM-MSC increased in vitro H9c2 cell viability after hypoxia incubation - Combined hAM-MSC transplantation produced ↑engraftment and ↑survival of MSCs versus isolated MSC transplantation - hAM-MSC versus isolated hAM versus isolated MSC: - hAM enhances the therapeutic benefit of MSC transplantation post-MI - ↑EF, ↑angiogenesis, ↓infarct size, ↓inflammatory infiltrate, ↓fibrosis - Improved myocardial tissue structure
Acellular hAM⁵²	Chronic HF: Permanent LAD ligation Epicardial application 7 days after LAD ligation (single dose)	- ↑EF but no significant difference in LVESV or LVEDV versus controls - ↑NFkB in hAM versus controls - No difference in TNF-a or ROS production - ↑NALP3, ↓IL-1B - No difference in ASC or Caspase-1
Acellular hAM⁴⁶	Chronic HF: Permanent RCA ligation Topical application 14 days after ligation	- nVCM “sandwich”: Decellularized myocardial tissue from human fetuses used as a scaffold which is coated with rat nVCMs and then covered with acellular hAM - hAM promotes cell viability with increased longevity of transplanted nVCMs when hAM is included in the scaffold - ↑angiogenesis, ↓fibrosis, ↓scar tissue - No functional or structural evaluation of myocardium
hAM-MSC-CM⁵³	Chronic HF: Isoproterenol injection Intramyocardial injection after HF phenotype development	- ↑EF, FS in CM treated animals - ↓Caspase-3 and 9 expression - CM appears to improve HF via modulation of apoptosis
Acellular hAF⁷	Ischemia-Reperfusion (60 min LAD ligation and release) Intramyocardial and intravenous injections 30 min after reperfusion	- ↑EF, ↓LVIDD for hAF treated animals - ↓Wet/dry lung weight ratio, ↓Heart/body weight ratio at 4 weeks - ↓ Infarct size/AAR, ↓serum cTnI at 24 h - ↓LV fibrosis - No evidence of inter-donor variability: Results preserved across 6 unique hAF donors
hAFMSC⁵⁴ (secretome)	Ischemia-Reperfusion (30 min LAD ligation and release) Injection via tail vein 5 min before reperfusion	- Secretome collection by in vitro hypoxia culture (30 h) of hAFMSCs - ↓Infarct size/AAR ratio - ↓Cardiomyocyte separation & inflammatory cell infiltrate, ↓Apoptosis - ↑Cell proliferation - ↑ Expression of angiogenesis & anti-inflammatory genes
hAF-MSC-EV⁸	Isoproterenol induced-cardiac fibrosis Injection via tail vein for 7 days (multiple doses)	- ↓Fibrosis, ECM deposition, collagen I - ↑Angiogenesis, CD31, micro-vessel density in micro-CT scanning
hAECs and hAEC-EV¹¹	Acute MI without reperfusion Intramyocardial injection following LAD ligation	- ↑EF, ↓LVEDD in EV and hAEC-treated hearts - ↓Fibrosis and apoptosis in EV and hAEC-treated groups - ↑HUVEC tubule formation in EV and hAEC-exposed cells
hAMSC⁵⁵ (secretome)	Ischemia-Reperfusion (30 min LAD ligation and release) hAMSC-CM injection after 15 min reperfusion	- hAMSC-CM ↓cTn-I and MDA levels and ↑SOD and GPx activities - hAMSC- CM improved cardiac histological changes and decreased myocardial injury percentage
hAFS-EV¹⁰	Ischemia-Reperfusion (30 min LAD ligation and release) Jugular vein injection 2 min before reperfusion	- SEC-purified small EVs, secreted from hAFS in vitro culture -↓Infarct size/AAR ratio - Proteomic analysis: angiogenesis, cardio-vascular development, promigratory
hAFS and hAFS-EV⁵⁶ (secretome)	Acute MI without reperfusion (Permanent ligation of LAD) Intramyocardial injection	- Secretome obtained by hypoxic culture of hAFS, EVs by serial ultracentrifugation - ↓Apoptosis, ↓Acute inflammatory response (MPO + neutrophils, CD68 + macrophages, IL-1α, MPO expression) at 24 h - ↑EF (in both hAFS secretome and EV treated groups) at 7 & 28 days - ↑Density of microvessel (PECAM-1+) and arterioles (SMA+) - ↑Cell-cycle re-entry - ↑CPC activation, at 3 h after injection, ↑EPDC activation - No effect by hAFS-CM (secretome) depleted of EVs
hAFS-CM¹³ (secretome)	mNVCM and H9C2 cells exposed to Doxorubicin	- hAFS secretome rescues H9c2 cells from Dox-induced senescence and apoptosis - hAFS-CM prevents mNVCM from Dox toxicity via NFkB pro-survival genes Il6 and Cxcl1 and the PI3 K/Akt pathway - hAFS-CM antagonizes Dox toxicity on human cardiac progenitor cells
hAMSC-CM⁴⁵ (secretome)	Ischemia-Reperfusion (30 min LAD ligation and release) hAMSC-CM injection after 10 min reperfusion	- In-vitro: cytoprotective and proangiogenic - In-vivo: ↓infarct size, ↓cardiomyocyte apoptosis, ↓ventricular remodeling, ↑capillary formation at infarct border zone - Identified 32 genes encoding secreted factors expressed by hAMSCs and found within their CM

Abbreviations: AAR, area at risk; AF-PCs, amniotic fluid-derived progenitor cells; AMI, acute myocardial infarction, ASC (Apoptosis-associated speck-like protein containing a caspase recruitment domain); CM, conditioned media; CPC, resident cardiac progenitor cells; cTnI, cardiac-specific Troponin I; cTnT, cardiac troponin T; Dox, Doxorubicin; ECM, extracellular matrix; EF, ejection fraction (%); EPDC, adult epicardium derived progenitor cells; EV, extracellular vesicles; FS, fractional shortening (%); GPx, glutathione peroxidase; hAFMSC, human amniotic fluid derived mesenchymal stem cells; hAM, human amniotic membrane; hAEC, human amniotic epithelial cell; hAMSC, human amniotic mesenchymal stromal cell; hAMSC-CM, human amniotic mesenchymal stromal cell conditioned media; hAFS, human amniotic fluid stem cell; HUVEC, human umbilical vein endothelial cells; IL, interleukin; IRI, ischemia-reperfusion injury; LAD, left anterior descending coronary artery; LV, left ventricle; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; MDA, malondialdehyde; mNVCM, mouse neonatal ventricular cardiomyocytes; MRI, magnetic resonance imaging; MSCs, mesenchymal stem cells, NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SD, Sprague-Dawley; SEC, size-exclusion chromatography; SOD, superoxide dismutase; TNF-a, tumor necrosis factor-alpha.

Description of preclinical studies related to the use of acellular components of human amniotic fluid in the treatment of cardiac disease. Some of the included studies did utilize cells in addition to extracellular vesicles or the secretomes of the cells and, as such, have been included for completeness. Studies are organized in descending chronological order.

Translation to human clinical experience remains limited. Two small studies describing human cardiac surgery subjects who received AM pericardial substitutes have demonstrated a reduction of postoperative inflammation (evidenced by reduced pericardial effusion) and atrial fibrillation, but the total number of included patients is too low to promote widespread implementation.^6,48 Most recently, a clinical trial in human cardiac surgery patients (NCT04130061: Human Amniotic Membrane to Decrease Post Operative Atrial Fibrillation) reported preliminary, unpublished results from 27 enrolled patients who received topical application of AM patches following cardiac surgery. There were no adverse events related to AM application and statistical analysis comparing effectiveness for anti-inflammation and atrial fibrillation prevention is ongoing.

Amnion-Derived Cells

Stem cells of various types have been extensively investigated in the field of regenerative medicine for their differentiation capacity; however, their clinical application has been limited. Adult stem cells (eg, bone marrow–derived, adipose-derived, skeletal muscle and resident cardiac stem cells) have the benefit of being autologous in nature, but have been ultimately hindered by (1) their limited ability to undergo “trans-differentiation” to assorted cell types, (2) prolonged time required for culture and expansion of meaningful numbers for transplantation, and (3) limited capacity for retention within injured myocardium.⁵⁷ Embryonic stem cells, unlike adult stem cells, possess a potent differentiation capacity, prolific growth in cell culture, and have demonstrated impressive results for myocardial regeneration in preclinical rodent experiments.⁵⁸ However, the substantial ethical concerns related to their use in addition to their immunogenicity and tumorigenicity have limited their clinical application.

Amniotic products, on the other hand, contain a heterogeneous population of cells that are readily obtained either during routine prenatal testing or at the time of delivery and have a high ethical acceptability, low immunogenicity and tumorigenicity, and a cellular potency that falls between that of embryonic and adult stem cells.^21,59

Human Amniotic Epithelial Cells

Human amniotic epithelial cells form the innermost layer of the amnion, lining the amniotic cavity in direct contact with the AF. Isolation typically occurs via enzymatic digestion of the AM after mechanical separation of the AM from the underlying chorion. Surface markers of the hAECs include embryonic-specific markers such as stage-specific antigens (SSEAs) 3 and 5, Tra-1-60, Tra-1-81, and mesenchymal markers CD105, CD90, CD73, CD44, CD29, HLA-A, -B, -C, CD13, CD10, CD166, and CD117.¹⁶ The hAECs are considered multipotent cells with the ability to differentiate into different lineages; however, they have also been shown to express the pluripotent stem cell–specific transcription factors octamer-binding protein 4 (Oct-4) and nanog with in vitro differentiation into all 3 germ layers.²¹

Clinical utilization of hAECs for the treatment of cardiac-specific disease in human patients has not been reported but has been explored in the preclinical setting with attempts to promote their differentiation into cardiomyocyte-like cells both in vitro and in vivo.^21,35 The hAECs have differentiated into cardiomyocyte-like cells in vivo following transplantation in a rat model of myocardial ischemia; however, the overall rate of differentiation was quite low (3% of transplanted cells expressed cardiac-specific genes and proteins). Nonetheless, the intervention resulted in a significant increase in cardiac function and reduction in infarct area compared to untreated controls. This led to the conclusion that it was likely not the differentiation or stem cell plasticity of the hAECs that were contributing to recovery, but rather a “trophic” effect of the transplanted cells on the proximate injured cells of the myocardium.³⁵ Subsequently, this group demonstrated that the cellular medium from hAECs cultured in hypoxic conditions was rich in 4 pro-angiogenic cytokines (angiogenin, epidermal growth factor, interleukin-6, and monocyte chemoattractant protein-1). They then further demonstrated that the transplantation of hAECs into infarcted rat myocardium 10 days after LAD ligation resulted in increased production of the same 4 cytokines in vivo as confirmed by the presence of human mRNA detected by RT-PCR using human primers, thus bolstering their previous argument for the presence of a paracrine effect on injured myocardium.⁶⁰ Similarly, more recent investigation in a rodent MI model utilized human amniotic epithelial stem cells (hAESCs) for isolated myocardial injection as well as implant within a polyurethane scaffold for topical application in the infarcted area. Treatment resulted in improved LV function and reduction in adverse remodeling with prevention of the development of a dilated cardiomyopathy phenotype (increased LVEF, reduced LVEDD). Subsequent in vitro exploration implicated the paracrine modulation of the microenvironment via secretion of factors with antioxidant, anti-inflammatory, ECM remodeling, and pro-angiogenesis properties, hinting at potential mechanisms for the observed cardioprotection.⁶¹ Once again, the differentiation of transplanted hESCs did not appear responsible for the protective benefits, but rather paracrine modulation of the environment seemed to play the dominant role.

Human Amniotic Mesenchymal Stromal Cells

Human amniotic mesenchymal stromal cells are multipotent cells capable of differentiation into mesoderm-derived (adipogenic, chondrogenic, myogenic, and osteogenic) lineages and are isolated from the AM deep to the hAECs. These cells are isolated after mechanical and chemical digestion using trypsin and collagenase to separate them from the overlying epithelium and the surrounding basement membrane of the mesenchymal layer of the AM, respectively.⁶² Cell surface markers are consistent with those reported for bone marrow–derived mesenchymal cells and include SH3, SH4, CD29, CD44, and CD166 while being negative for hematopoietic markers.^42,62 The immunologic tolerance, pro-angiogenic traits, high expansion potential, and ease of obtainment from what is otherwise considered a waste material (placenta), of hAMSCs have fueled several preclinical studies examining myocardial ischemia and its sequelae.⁶³ These studies have largely focused on the ability of hAMSCs to form endothelial cells, promote chemotaxis, and promote angiogenesis for the treatment of acute myocardial ischemia.^42,43,45 A systematic review and meta-analysis identified 3 preclinical studies utilizing hAMSCs (and 1 study using porcine AMSCs) for the treatment of myocardial ischemia and concluded that hAMSCs administration resulted in the reduction of postinfarction LV cavity dilation and improvement in LV systolic function in models of acute ischemia but was unable to provide significant data regarding the effect on congestive heart failure.^42,63 Similar to hAECs, no clinical trials of hAMSCs have been reported.

Amniotic Fluid-Derived Cells and Heart Disease

Human AF-Derived MSCs

Human AF-derived MSCs are readily obtained and isolated from human AF using various protocols that have been previously described. The hAFMSCs display remarkable expansion capability with rapid in vitro growth that is greater than that of bone marrow–derived MSCs. Despite their high rate of proliferation, they retain a normal karyotype and do not display tumorigenicity.¹⁶ Cell surface markers are positive for mesenchymal markers (CD90, CD73, CD105, CD166), adhesion molecules (CD29, CD44, CD49e, CD54), and antigens of MHC-1. They are negative for hematopoietic and endothelial markers, and they lack the markers of ESCs and other pluripotent cells such as Oct-4 and nanog. The hAFMSCs are multipotent with differentiation toward mesenchymal lineages, but have demonstrated the ability to undergo reprogramming into pluripotent stem cells with a greater reprogramming capacity and in less time than that required for somatic cells such as human fibroblasts.^4,16

Preclinical studies in cardiac disease have focused heavily on cellular therapy applications with the primary goal being regeneration of cardiac tissue following injury. Different groups have evaluated the regenerative potential of hAFMSCs with adequate demonstration of differentiation into cardiomyocytes in vitro, but when transplanted into injured myocardium, only limited differentiation of hAFMSCs into cardiomyocytes has been observed.^64,65 More frequently, the hAFMSCs undergo successful engraftment with differentiation into endothelial and smooth muscle cells with a smaller proportion differentiating into effective, contractile cardiomyocytes.⁶⁶ Interestingly, despite this limited myocardial regeneration in animal models of cardiac injury, cardiac function still improves following cellular transplantation.⁶⁷ Consequently, it has been speculated that this improvement may be related more to a paracrine influence of the transplanted cells rather than tissue regeneration.

Clinical experience with hAFMSCs is minimal. A single clinical case report published in 2018 describes the implantation of hAFMSCs within a micronized human allograft-derived liquid matrix in a hybrid approach in conjunction with transmyocardial revascularization for the treatment of a patient with refractory angina and ischemic cardiomyopathy. Their experience describes postprocedural resolution of cardiac symptoms and functional improvement which was corroborated by noninvasive testing of myocardial viability biomarkers via cardiac magnetic resonance imaging and thallium imaging, demonstrating ventricular remodeling in a region of infarct.⁶⁸ Notably, there was no histological assessment or other means of verification of the ultimate fate of the transplanted hAFMSCs nor was there any control for comparison of the impact without hAFMSC transplant. Therefore, it remains unclear whether the benefit in this single case report derives from the hAFMSCs or the laser revascularization; no subsequent experience has been reported.

Human AF Stem Cells

Human AF Stem Cells (hAFS) are a select population of AF-derived cells (∼0.5%-1.0%) with stem cell potential as determined by the expression of markers consistent with a pluripotent undifferentiated state such as Oct-4 and nanog.¹⁸ Most commonly, these cells are being isolated and selected via CD-117 (c-Kit) positivity in a 2-step protocol consisting of immunological selection of c-kit positive cells and subsequent expansion in vitro. The hAFS cell surface markers are notable for the presence of both ESC (SSEA-4) and mesenchymal cell (CD73, CD90, CD105) markers, adhesion molecules (CD29, CD44), and antigens in the MHC-I family. They are negative for hematopoietic and endothelial markers and, notably, antigens of MHC-II.^16,18 The hAFS have a high proliferation rate and derived clonal cell lines differentiate into tissues of all 3 germ layers spontaneously. This capacity for rapid expansion and their minimal immunogenic profile, broad multipotency, and lack of tumor formation in vivo has led to their application in cellular therapy and tissue engineering.^16,18,69

Attempts to promote in vitro cardiac differentiation have yielded mixed results. Various techniques have been employed including coculture with neonatal rat ventricular myocytes,^70,71 the use of various differentiation media,^65,70 and treatment with 10 uM 5-aza-2′deoxycytidine for 24 h.⁷² Successful cardiogenic lineage differentiation has been achieved and verified by cardiac troponin I expression as well Connexin 43 gap junction formation and localization to the cellular membrane; however, generation of spontaneously beating and contractile cardiomyocytes has not been successful in unaltered hAFS (some success in hAFS that have undergone induced pluripotency).⁷³ Consequently, enthusiasm for hAFS regeneration of viable myocardium has waned, but this is not to say that hAFS therapy has been fruitless. Injection of hAFS cells into infarcted rodent myocardium has reduced fibrosis and LV enlargement,^71,74,75 increased myocardial vascular density,^74,75 reduced infarct size,²⁶ and produced significant improvement in contractile function compared to saline-injected controls.^74,75 These results are observed despite only modest differentiation, proliferation, and retention of hAFS within the infarcted rodent myocardia. As a result, and consistent with other cell therapy studies, investigators have speculated that the witnessed recovery of cardiac function may be independent of hAFS cellular incorporation, and, more likely, is related to paracrine modulation via soluble bioactive factors (subsequently referred to as the “secretome”).^26,54,56,76

Studies from the early 2010s illuminated the potential paracrine impacts of the hAFS. In 2016, it was demonstrated that mouse neonatal ventricular cardiomyocytes (mNVCMs) exposed to the secretome isolated from hypoxic-preconditioned hAFS protected against Doxorubicin-induced cellular senescence and apoptosis through the increased expression of cytokines, chemokines, growth factors, and their respective receptor binding proteins on mNVCMs.¹³ Others reproduced this protection against cardiac reperfusion injury in mouse models and in vitro studies of human cardiomyocyte proliferation (both under normoxic and hypoxic conditions) following administration of the secretome of hAFS that had been cultured in hypoxia (1% O2) for 30 h.⁵⁴ More recent characterization of the hypoxia-augmented hAFS secretome has demonstrated the increased presence of pro-angiogenic and anti-inflammatory factors, fueling the pursuit of further refinement of the hAFS secretome for treatment of ischemic cardiac injury.

With respect to tissue engineering, hAFS have been posited as an ideal cell type for the fabrication of valve leaflets. Clinically, the proposed treatment pathway would be: prenatal identification and characterization of congenital defect, imaging-based 3-D reconstruction of defect with subsequent creation of an individualized polymer valve scaffold, prenatal harvest of autologous hAFS via amniocentesis, in vitro seeding and subsequent growth of hAFS seeded valve leaflets, and ultimately, implantation of the autologous neo-tissue valve which would then grow with the patient. Preclinical work has demonstrated the feasibility of the creation of hAFS-derived autologous valves with similar chemical, biological, and mechanical properties compared to native valves.^77–79 Clinical implementation in human patients has not yet been realized, but a proof-of-concept study demonstrated the in vivo functionality of minimally invasive in utero implantation of an autologous tissue-engineered heart valve.⁷⁸

Nonetheless, clinical use of hAFS is limited to a single case report in which a patient with refractory cardiogenic shock was unloaded with an extracorporeal left ventricular assist device and received concomitant intramyocardial injection of hAFS at the left ventricular apex, lateral inferior walls, and intravenous injection into the right subclavian vein. The patient was weaned off mechanical support after roughly 3 weeks and was ultimately discharged to a rehabilitation facility.⁵ As with the other singular case reports, there is insufficient evidence to support widespread clinical utilization of hAFS in a similar approach.

Acellular Amniotic Products and Heart Disease

The recognition of a beneficial paracrine influence during early amniotic cellular therapy led investigators to consider the role of the acellular components of the amnion. More specifically, the secretome of amniotic cells, the acellular AF, and the exosomes contained therein, have inspired deeper investigation. Table 1 provides an overview of the studies pertaining to these acellular amniotic products in the treatment of heart disease.

Exosomes/EVs

Exosomes are small anucleic EVs with a bilayer membranous structure and a diameter of 30 to 150 nm that contain various RNAs, proteins, DNA, and lipids.⁹ Initial preclinical EV studies demonstrated that EVs isolated from adult MSCs were cardioprotective against IR injury in a mouse model of acute MI with reduction in infarct area and evidence of pro-angiogenesis and antiapoptosis.¹² Subsequent studies using amniotic-derived cells and exosomes from both AM (hAECs) and AF (hAFMSCs) have shown the ability to reduce myocardial ischemia-reperfusion injury, attenuate oxidative stress, and inhibit apoptosis as well as enhance angiogenesis and stimulate endogenous cardiac progenitor cell activation to foster cardiac repair and regeneration.^10,11 Additionally, amniotic-derived exosomes have shown promise in their ability to modulate cardiac fibrosis via reduction in fibroblast activation and collagen synthesis.⁸ In short, these data inspire not only hope for the eventual translation of amniotic-derived EVs into clinical practice but also present challenges related to the standardization of administration and optimization of isolation and characterization specificity.

Acellular AF

Human AF contains a host of cellular and acellular elements that can reduce inflammation, possess antimicrobial properties, and carry a low risk of immunogenicity.^80–82 We have developed a proprietary method whereby allogenic human AF (hAF) is procured and processed from volunteer donors at the time of elective cesarean section. When filtered and sterile-processed, hAF can be rendered devoid of insoluble, cellular products (ie, cells, vernix, and lanugo) such that administration is without risk of AF embolism.⁸³ Purified hAF is a nonantigenic solution of EVs, hundreds of naturally derived proteins such as hyaluronic acid and a host of cytokines and organic and inorganic compounds.^81,83

We recently investigated the therapeutic role of the acellular hAF in a rat model of ischemia-reperfusion injury. Compared to animals that received saline, hAF animals demonstrated preservation of ventricular function and marked reduction in infarct area, fibrosis, cytokine expression, and inflammatory cell infiltration.⁷ The hAF is rich in proteins that relate to host defense and when further subcategorized are associated with the inflammatory response, innate immunity, cell adhesion, proliferation, and angiogenesis (Figure 2).^7,81 While a specific mechanism of action remains unknown, it seems likely that immunomodulation and attenuation of the inflammatory response, among several other targets, are central to its efficacy (Figure 3).

Conclusion

Despite decades of effort and the identification and implication of numerous processes in the development of cardiovascular pathologies (such as cell death or inflammatory signal transduction), targeted interventions on specific mechanistic pathways often prove worthy in preclinical models, yet struggle in clinical translation.^84–89 While reasons for this observation are certainly multifactorial, one possible explanation may be that attempts to simplify an otherwise complex and diverse biologic system into a distillate that focuses on a particular receptor, protein, or family of transcription factors are inherently short-sighted. Such singular, targeted therapies do not account for intrinsic autoregulatory feedback loops, compensatory adaptation, and the myriad of other pathways intertwined in constant flux—all of which can ultimately conspire to blunt the clinical applicability of a highly specific objective. One of the attractive features of utilizing amniotic products for the treatment of heart disease is that it does not rely on one pathway or a single mechanism to impart its effect. For example, acellular hAF is chock-filled with thousands of proteins and insoluble compounds, some at barely detectable levels that no drug company could possibly recreate. Indeed, the amnion and its products are evolutionarily derived over millennia, house nature's own pharmacy, and deserve to be further explored in their application to treat cardiovascular disease.

Footnotes

Acknowledgments

This research was supported in part by internal funding from the University of Utah Division of Cardiothoracic Surgery, American Heart Association Strategic Focused Research Network—Heart Failure, the Nora Eccles Treadwell Foundation (CHS), and the National Institutes of Health under Ruth L. Kirschstein National Research Service Award T32HL007576-37 from the National Heart, Lung, and Blood Institute (IN). Figure 3 was created in BioRender. Nickel, I. (2025)

ORCID iD

Ian Nickel

Author Contributions

Ian Nickel, Grace Mitchell, Hadi Javan, Carla Valenzuela Ripoll, Jan Pierce, and Craig H. Selzman contributed to the review process and writing of the manuscript; Craig H. Selzman provided oversight and editing of the manuscript; all authors approved the final version of the manuscript.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

One author (JP) holds a patent related to the processing and manufacturing of acellular human amniotic fluid. The other author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

The data from which this review paper is derived are publicly available and have been referenced/cited where appropriate.

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