Amniotic Fluid Stem Cells and Their Secretomes as tools of regenerative medicine; Influence of Donor Characteristics on Standardization

Amniotic fluid has emerged as a promising source of stem cells for tissue regeneration. Ethical concerns surrounding the isolation of amniotic fluid are minimal, as it can be safely collected during routine procedures such as second trimester amniocentesis, third trimester amnioreduction, or caesarean section at the end of gestation ^[1]. Amniotic fluid cells exhibit heterogeneity in morphology, in vitro, and in vivo characteristics, derived mainly from fetal tissues such as skin, respiratory, intestinal, and urinary tracts, as well as the amniotic membranes and connective tissues. Various cellular subpopulations exist within amniotic fluid, including epithelioid, “amniotic,” and fibroblastic types, whose proportions vary according to gestational age. Amniotic fluid mesenchymal stem cells (AFMSC) are particularly promising due to their therapeutic potential which have been tested in different studies of muscle, cartilage, and bone defects ^[1]. The term “secretome” was initially introduced by Tjalsma et al., defining it “both the components of machineries for protein secretion and the native secreted proteins”. This definition underwent further refinement by Hathout and Agrawal, who elaborated it to encompass factors secreted by cells, tissues, or organisms into the extracellular space under defined temporal and environmental conditions. Presently, it has been recognized that the secretome comprises not only soluble factors but also includes lipids and extracellular vesicles (EVs) containing vital molecules ^[2]. This novel perspective on cell-free-based therapies, which focuses on harnessing the secretome of cells, gained prominence inspiring the exploration of secretome-based applications across various domains of regenerative medicine. Due to concerns regarding potential adverse effects associated with cell-based therapies, including uncertainty regarding long-term recipient outcomes, there has been growing interest in the secretome produced by cells which have been attributed to their regenerative effects per se, particularly the secretome derived from Mesenchymal stem cells (MSCs). Utilization of MSC secretome offers several significant advantages, including the avoidance of invasive procedures for cell extraction, the ability to conduct pharmacological dose and safety assessments, ease of application, and the potential for manipulation of its composition ^[3]. Furthermore, MSC-derived secretome pivotal role in modulating innate and adaptive immune responses underlie their sought-after anti-inflammatory properties ^[3].

In the latest issue of JSRM, Klaymook et al. ^[4] have elucidated the therapeutic effects and underlying mechanisms of the amniotic fluid-derived mesenchymal stem cell (AF-MSC) secretome (AFS-se) in Osteoarthritis (OA). Their study reveals that AFS-se exerts therapeutic effects on OA by suppressing the inflammatory actions of IL-1β and TNF-a through protein phosphorylation within the MAPK pathway, while simultaneously enhancing the regenerative and self-repair functions of chondrocyte progenitor cells (CPCs), even within traumatized cartilage.

While this endeavor is laudable in harnessing the valuable source of MSC secretome found in amniotic fluid, typically discarded as waste, further advancements can be made by standardizing the sourcing of amniotic fluid and validating donor characteristics to ensure the selection of appropriate amniotic fluid MSCs. Various donor-specific factors such as age, sex, alcohol and/or tobacco consumption, and comorbidities such as obesity or diabetes have been identified in the literature to contribute to inter-individual variability in pooled MSCs through complex mechanisms. For instance, immunosuppressive capacities have been linked to donor weight, as adipose stem cells (ASCs) from heavier twins exhibited significantly higher TNF expression, lower angiogenic potential, and greater immunosuppressive capacity compared to leaner cotwins ^[5].

In the case of amniotic fluid-derived stem cells and their secretome, it is proposed that additional factors need consideration, including maternal age at conception, presence or absence of gestational diabetes, presence of genetic abnormalities in the offspring, and notably, gut microbiome composition. Modulation of gut microbiota through antibiotic treatments in conventional mice or administration of specific gut microbiota in germ-free mice has demonstrated effects on bone mass and growth, mediated through commensal bacteria-secreted short-chain fatty acids that regulate skeletal growth hormones, such as insulin-like growth factor ^[6]. Additionally, disruption of the microbiota has been implicated in perturbing the normal circadian clock, potentially exacerbating metabolic disorders and aging-related declines in stem cell function and pool exhaustion ^[7]. The gut microbiome has also emerged as a pivotal element in regenerative medicine. It comprises a diverse array of cells and factors that not only contribute to cell differentiation, proliferation, and paracrine signalling but also exert profound effects on tissue repair and regeneration, even remotely, through inter-organ axes such as the gut–brain, gut–lung, and gut–skin axis ^[8]. Exploring the gut microbiome's impact on amniotic fluid stem cells, particularly concerning misfolded proteins, holds promise for identifying potential neurodegenerative disorders in the fetus ^[9]. Therefore, in harnessing amniotic fluid stem cells and their secretome, assessing the influence of the gut microbiome in both the mother and fetus is imperative for obtaining high-quality stem cells. Furthermore, the timing of AFSC collection is crucial concerning genetic abnormalities in offspring, as chemical reprogramming is more easily achieved in first-trimester AFSCs, while successful reprogramming in second-trimester AFSCs necessitates prior exposure to embryonic-type culture conditions. Previous studies have observed no irregularities in the epigenetic control system in early-passage AFSCs ^[1]. Amniotic fluid also serves as a reflection of both maternal and fetal health. Its composition, including carbohydrates, lipids, phospholipids, urea, and proteins, dynamically changes throughout pregnancy, providing valuable insights into fetal and maternal well-being ^[10–12]. Amniotic fluid analysis can offer information on fetal maturation, particularly the development of lungs and kidneys, as well as facilitate biochemical exchanges ^[11]. Before 20 weeks of gestation, the non-keratinized fetal skin enables bidirectional transport of soluble molecules between the amniotic fluid and fetal structures. Moreover, the presence of cells from various fetal organs in amniotic fluid aids in diagnosing fetal malformations, chromosomal diseases, and other conditions. Additionally, amniotic fluid analysis can detect infections and assess the immune status of both the mother and fetus ^[12]. Therefore, in utilizing amniotic fluid-derived MSCs and their secretome for regenerative medicine, comprehensive analysis of maternal and fetal health is imperative.

As evidence on the potentials of human amniotic fluid and their secretome are pointing to their potential in several regenerative medicine applications, further research in standardizing their characteristics based on donor specific characteristics of both mother and the fetus described above, could help establish standardized criteria for donor-derived AFSCs to be used in regenerative medicine applications.