Intermembrane distances at the feto-maternal interface in epitheliochorial placentation.

INTRODUCTION: In an epitheliochorial placenta, the apical membranes of trophoblast cells and of uterine epithelial cells are in contact to each other (feto-maternal contact). In addition, there are also folds in which the trophoblast membrane is in contact with itself (feto-fetal contact) and areas where apical uterine epithelial membrane is in contact with itself (materno-maternal contact). METHODS: We use transmission electron microscopy of placental samples from pigs. (n = 3), cows (n = 2), sheep (n = 2), goat (n = 2) and roe deer (n = 1) to study the intermembrane distance in these three contact types. RESULTS: The measured intermembrane distances vary between 8 and 25 nm. One common feature is that the distance at feto-fetal contact sites is about 6-10 nm wider than at materno-maternal sites and feto-maternal sites show intermediate values. DISCUSSION: This finding suggests that the membrane distance at feto-maternal contact sites is determined by heterophilic binding of larger fetal to smaller maternal binding molecules. Homophilic binding of smaller maternal or larger fetal molecules lead to the smaller or wider intermembrane distances at materno-maternal or feto-fetal contact sites respectively. The observation that this similar pattern of membrane distances is present in pigs and in ruminants suggest that an evolutionary mechanism is involved in determining the intermembrane distance in epitheliochorial placentas.

Sustained intrinsic WNT and BMP4 activation impairs hESC differentiation to definitive endoderm and drives the cells towards extra-embryonic mesoderm.

We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain endoderm derivatives, differentiating instead into extra-embryonic mesoderm. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of endoderm differentiation and induction of extra-embryonic mesoderm markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.

Polymicrobial stimulation of human fetal membranes induce neutrophil activation and neutrophil extracellular trap release.

Preterm birth is a major contributor to neonatal mortality and morbidity. While the causes of preterm birth remain incompletely understood, infection is a major risk factor, and chorioamnionitis is commonly observed. Chorioamnionitis is characterized by inflammation and neutrophil infiltration of the fetal membranes (FM). We recently reported that human FMs which had been exposed to low levels of bacterial lipopolysaccharide (LPS) recruit neutrophils and activate them, increasing their secretion of pro-inflammatory cytokines, degranulation of myeloperoxidase (MPO), and release of neutrophil extracellular traps (NETs). Herein, we demonstrate that conditioned media (CM) from viral dsRNA (Poly(I:C))-stimulated FMs also increased neutrophil migration, and induced the secretion of inflammatory IL-8 and the release of NETs. Furthermore, CM from FMs stimulated by a combination of bacterial LPS and Poly(I:C) augmented neutrophil NET release, compared to CM from FMs stimulated with either Poly(I:C) or LPS alone. NETs induced by FMs exposed to Poly(I:C), with or without LPS, were released and degraded quicker than those induced by resting or LPS-stimulated FM-CM. These findings indicate that FMs exposed to viral dsRNA promote neutrophil recruitment, activation and NET formation, similar to FMs exposed to bacterial LPS alone. However, in response to FM polymicrobial stimulation the levels and kinetics of NET release are augmented. This work builds upon our understanding of how infections at the maternal-fetal interface may affect neutrophil function.

Reassembling gastrulation.

During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important prerequisite for the emergence of patterns within the developing organism is to establish asymmetries at various scales, ranging from individual cells to the entire embryo, eventually giving rise to the different body structures. This becomes especially apparent during gastrulation, when the earliest major lineage restriction events lead to the formation of the different germ layers. Traditionally, the unfolding of the developmental program from symmetry breaking to germ layer formation has been studied by dissecting the contributions of different signaling pathways and cellular rearrangements in the in vivo context of intact embryos. Recent efforts, using the intrinsic capacity of embryonic stem cells to self-assemble and generate embryo-like structures de novo, have opened new avenues for understanding the many ways by which an embryo can be built and the influence of extrinsic factors therein. Here, we discuss and compare divergent and conserved strategies leading to germ layer formation in embryos as compared to in vitro systems, their upstream molecular cascades and the role of extrinsic factors in this process.

Characterizing the immune cell population in the human fetal membrane.

PROBLEM: This study localized CD45+ immune cells and compared changes in their numbers between term, not in labor (TNIL) and term, labor (TL) human fetal membranes. METHOD OF STUDY: Fetal membranes (amniochorion) from normal TNIL and TL subjects were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry for evidence of total (CD45+ ) immune cells as well as innate immune cells (neutrophils, macrophages and NK cells) using specific markers. Fetal origin of immune cells was determined using polymerase chain reaction (PCR) for SRY gene in Y chromosome. RESULTS: CD45+ cells were localized in human fetal membranes for both TNIL and TL. A threefold increase in CD45+ cells was seen in TL fetal membranes of (7.73% ± 2.35) compared to TNIL (2.36% ± 0.78). This increase is primarily contributed by neutrophils. Macrophages and NK cells did not change in the membranes between TNIL and TL. Leukocytes of fetal origin are present in the fetal membranes. CONCLUSION: The fetal membranes without decidua contain a small proportion of immune cells. Some of these immune cells in the fetal membrane are fetal in origin. There is a moderate increase of immune cells in the fetal membranes at term labor; however, it is unclear whether this is a cause or consequence of labor. Further functional studies are needed to determine their contribution to membrane inflammation associated with parturition.

Current Status and Future Prospects of Perinatal Stem Cells.

The placenta is a temporary organ that is discarded after birth and is one of the most promising sources of various cells and tissues for use in regenerative medicine and tissue engineering, both in experimental and clinical settings. The placenta has unique, intrinsic features because it plays many roles during gestation: it is formed by cells from two individuals (mother and fetus), contributes to the development and growth of an allogeneic fetus, and has two independent and interacting circulatory systems. Different stem and progenitor cell types can be isolated from the different perinatal tissues making them particularly interesting candidates for use in cell therapy and regenerative medicine. The primary source of perinatal stem cells is cord blood. Cord blood has been a well-known source of hematopoietic stem/progenitor cells since 1974. Biobanked cord blood has been used to treat different hematological and immunological disorders for over 30 years. Other perinatal tissues that are routinely discarded as medical waste contain non-hematopoietic cells with potential therapeutic value. Indeed, in advanced perinatal cell therapy trials, mesenchymal stromal cells are the most commonly used. Here, we review one by one the different perinatal tissues and the different perinatal stem cells isolated with their phenotypical characteristics and the preclinical uses of these cells in numerous pathologies. An overview of clinical applications of perinatal derived cells is also described with special emphasis on the clinical trials being carried out to treat COVID19 pneumonia. Furthermore, we describe the use of new technologies in the field of perinatal stem cells and the future directions and challenges of this fascinating and rapidly progressing field of perinatal cells and regenerative medicine.

Fetal Membrane Organ-On-Chip: An Innovative Approach to Study Cellular Interactions.

OBJECTIVE: Fetal membranes, a vital component that helps maintain pregnancy and contribute to parturition signaling, are often studied in segments due to its structural complexity. Transwells are traditionally used to study cell interactions; however, their usefulness is limited. To overcome these difficulties, a fetal membrane-organ-on-chip (FM-OO-C) was created to study interactive properties of amnion epithelial cells (AECs) and decidual cells compared to transwell systems. METHODS: Primary AECs and decidual cells from term, nonlaboring fetal membranes were cultured in a 2-chamber (AEC/decidual cell) FM-OO-C device and sandwiched between a semipermeable membrane. Cells were treated with cigarette smoke extract (CSE) or dioxin, and membrane permeability and cellular senescence were measured after 48 hours. The same experiments were conducted in transwells for comparisons. RESULTS: Compared to transwell cultures, FM-OO-C model produced better membrane permeability readings regardless of the side of treatment or time point. Membrane permeabilization was higher in AECs directly treated with CSE (1.6 fold) compared to similar treatment on the decidual side (1.2 fold). In FM-OO-C, treatments forced changes between cellular layers. This was evident when CSE and dioxin-induced senescence on one side of the chamber produced similar changes on the opposite side. This effect was minimal in the transwell system. CONCLUSION: The controlled environment of an FM-OO-C allows for improved signal propagation between cells by minimizing noise and highlighting the small changes between treatments that cannot be seen in conventional transwell devices. Fetal membrane-organ-on-chip provides a better interaction between cell types that can be used to study fetal-maternal signaling during pregnancy in future studies.

The domesticated buffalo - An emerging model for experimental and therapeutic use of extraembryonic tissues.

Large animals play important roles as model animals for biomedical sciences and translational research. The water buffalo (Bubalus bubalis) is an economically important, multipurpose livestock species. Important assisted reproduction techniques, such as in vitro fertilization, cryo-conservation of sperm and embryos, embryo transfer, somatic cell nuclear transfer, genetic engineering, and genome editing have been successfully applied to buffaloes. Recently, detailed whole genome data and transcriptome maps have been generated. In addition, rapid progress has been made in stem cell biology of the buffalo. Apart from embryonic stem cells, bubaline extra-embryonic stem cells have gained particular interest. The multipotency of non-embryonic stem cells has been revealed, and their utility in basic and applied research is currently investigated. In particular, success achieved in bubaline extra-embryonic stem cells may have important roles in experimental biology and therapeutic regenerative medicine. Progress in other farm animals in assisted reproduction techniques, stem cell biology and genetic engineering, which could be of importance for buffalo, will also be briefly summarized.

IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford.

The British Society for Developmental Biology Autumn Meeting, held in Oxford in September 2018, was the third in a series of international workshops which have been focussed on development at the extraembryonic-embryonic interface. This workshop, entitled "Embryonic-Extraembryonic Interactions: from Genetics to Environment" built on the two previous workshops held in 2011 (Leuven, Belgium) and 2015 (Göttingen, Germany). This workshop brought together researchers utilising a diverse range of organisms (including both vertebrate and invertebrate species) and a range of experimental approaches to answer core questions in developmental biology. This meeting report highlights some of the major themes emerging from the workshop including an evolutionary perspective as well as recent advances that have been made through the adoption of emerging techniques and technologies.

Expression and function of macrophage-inducible C-type lectin (Mincle) in inflammation driven parturition in fetal membranes and myometrium.

The pivotal role of inflammatory processes in human parturition is well known, but not completely understood. We have performed a study to examine the role of macrophage-inducible C-type lectin (Mincle) in inflammation-associated parturition. Using human samples, we show that spontaneous labour is associated with up-regulated Mincle expression in the myometrium and fetal membranes. Mincle expression was also increased in fetal membranes and myometrium in the presence of pro-labour mediators, the proinflammatory cytokines interleukin (IL)-1B and tumour necrosis factor (TNF), and Toll-like receptor (TLR) ligands fsl-1, poly(I:C), lipopolysaccharide (LPS) and flagellin. These clinical studies are supported by mouse studies, where an inflammatory challenge in a mouse model of preterm birth increased Mincle expression in the uterus. Importantly, elimination of Mincle decreased the effectiveness of proinflammatory cytokines and TLR ligands to induce the expression of pro-labour mediators; namely, proinflammatory cytokines and chemokines, contraction-associated proteins and prostaglandins, and extracellular matrix remodelling enzymes, matrix metalloproteinases. The data presented in this study suggest that Mincle is required when inflammatory activation precipitates parturition.

Maternal and fetal intrauterine tissue crosstalk promotes proinflammatory amplification and uterine transition†.

Birth is a complex biological event requiring genetic, cellular, and physiological changes to the uterus, resulting in a uterus activated for completing the physiological processes of labor. We define the change from the state of pregnancy to the state of parturition as uterine transitioning, which requires the actions of inflammatory mediators and localized paracrine interactions between intrauterine tissues. Few studies have examined the in vitro interactions between fetal and maternal gestational tissues within this proinflammatory environment. Thus, we designed a co-culture model to address this gap, incorporating primary term human myometrium smooth muscle cells (HMSMCs) with human fetal membrane (hFM) explants to study interactions between the tissues. We hypothesized that crosstalk between tissues at term promotes proinflammatory expression and uterine transitioning for parturition. Outputs of 40 cytokines and chemokines encompassing a variety of proinflammatory roles were measured; all but one increased significantly with co-culture. Eighteen of the 39 cytokines increased to a higher abundance than the sum of the effect of each tissue cultured separately. In addition, COX2 and IL6 but not FP and OXTR mRNA abundance significantly increased in both HMSMCs and hFM in response to co-culture. These data suggest that synergistic proinflammatory upregulation within intrauterine tissues is involved with uterine transitioning.

Amnion-Analogous Medical Device for Fetal Membrane Healing: A Preclinical Long-Term Study.

Although recent invasive fetal surgeries have improved fetal outcomes, fetal membrane rupture remains a major complication, leading to premature delivery, thus undermining the complete benefits of such procedures. A biocompatible amnion-analogous medical device (AMED) consisting of polycaprolactone framework and decellularized amniotic membrane (dAM)-derived hydrogel for restoration of amniotic membrane defect is developed using 3D printing technology. Its efficacy on healing iatrogenic fetal membrane defects in vitro is evaluated, showing that the dAM gel contains migratory and proliferative properties. The fetoscope feasibility of the developed AMED is assessed using a pregnant swine model. All animals had successfully recovered from anesthesia and the fetoscopic procedure and maintained a healthy condition until the end of the pregnancy. AMED exhibits superior surgical handling characteristics and is easy to manufacture, nonimmunogenic, biocompatible, and suitable for storage and transport for off-the-shelf use; hence, it can be used in successfully sealing defect sites, thus improving the preservation of the amniotic fluid, which in turn improves fetal survival and development.

Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols.

OBJECTIVE: To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue. RESULTS: A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol. CONCLUSIONS: The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.

Infection-induced thrombin production: a potential novel mechanism for preterm premature rupture of membranes (PPROM).

BACKGROUND: Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. Ureaplasma parvum is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. OBJECTIVE: This study was designed to investigate whether Ureaplasma parvum exposure can induce prothrombin production in fetal membranes cells. STUDY DESIGN: Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live Ureaplasma parvum (1×105, 1×106 or 1×107 colony-forming units per milliliter) or lipopolysaccharide (Escherichia coli J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ± standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett's test. RESULTS: Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by Ureaplasma parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, Ureaplasma parvum at a dose of 1×107 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. Ureaplasma parvum at a dose of 1×106 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). CONCLUSION: Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of Ureaplasma parvum-induced rupture of fetal membranes.

Classics revisited: Miguel Fernández on germ layer inversion and specific polyembryony in armadillos.

BACKGROUND: Miguel Fernández was an Argentinian zoologist who published the first account of obligate polyembryony in armadillos. His contribution is here discussed in relation to his contemporaries, Newman and Patterson, and more recent work. FINDINGS: Fernandez worked on the mulita (Dasypus hybridus). He was able to get early stages before twinning occurred and show it was preceded by inversion of the germ layers. By the primitive streak stage there were separate embryonic shields and partition of the amnion. There was, however, a single exocoelom and all embryos were enclosed in a common set of membranes comprising chorion towards the attachment site in the uterine fundus and inverted yolk sac on the opposite face. He showed that monozygotic twinning did not occur in another armadillo, the peludo (Chaetophractus villosus). CONCLUSIONS: Fernández's work represented a major breakthrough in understanding how twinning occurred in armadillos. His work and that of others is of intrinsic interest to zoologists and has a direct bearing on the origin of monozygotic twins and birth defects in humans.

Fetal Membranes-Derived Stem Cells Microenvironment.

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

Concise Review: Fetal Membranes in Regenerative Medicine: New Tricks from an Old Dog?

The clinical application of the fetal membranes dates back to nearly a century. Their use has ranged from superficial skin dressings to surgical wound closure. The applications of the fetal membranes are constantly evolving, and key to this is the uncovering of multiple populations of stem and stem-like cells, each with unique properties that can be exploited for regenerative medicine. In addition to pro-angiogenic and immunomodulatory properties of the stem and stem-like cells arising from the fetal membranes, the dehydrated and/or decellularized forms of the fetal membranes have been used to support the growth and function of other cells and tissues, including adipose-derived mesenchymal stem cells. This concise review explores the biological origin of the fetal membranes, a history of their use in medicine, and recent developments in the use of fetal membranes and their derived stem and stem-like cells in regenerative medicine. Stem Cells Translational Medicine 2017;6:1767-1776.

Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State.

Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state.

Generation of Canine Induced Extraembryonic Endoderm-Like Cell Line That Forms Both Extraembryonic and Embryonic Endoderm Derivatives.

Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.