Mesenchymal Stem/Stromal Cells and Their Role in Oxidative Stress Associated with Preeclampsia.

Preeclampsia (PE) is a serious medically important disorder of human pregnancy, which features de novo pregnancy-induced hypertension and proteinuria. The severe form of PE can progress to eclampsia, a convulsive, life-threatening condition. When placental growth and perfusion are abnormal, the placenta experiences oxidative stress and subsequently secretes abnormal amounts of certain pro-angiogenic factors (eg, PlGF) as well as anti-angiogenic factors (eg, sFlt-1) that enter the maternal circulation. The net effect is damage to the maternal vascular endothelium, which subsequently manifests as the clinical features of PE. Other than delivery of the fetus and placenta, curative treatments for PE have not yet been forthcoming, which reflects the complexity of the clinical syndrome. A major source of reactive oxygen species that contributes to the widespread maternal vascular endothelium damage is the PE-affected decidua. The role of decidua-derived mesenchymal stem/stromal cells (MSC) in normotensive and pathological placenta development is poorly understood. The ability to respond to an environment of oxidative damage is a "universal property" of MSC but the biological mechanisms that MSC employ in response to oxidative stress are compromised in PE. In this review, we discuss how MSC respond to oxidative stress in normotensive and pathological conditions. We also consider the possibility of manipulating the oxidative stress response of abnormal MSC as a therapeutic strategy to treat preeclampsia.

Human Decidua Basalis mesenchymal stem/stromal cells reverse the damaging effects of high level of glucose on endothelial cells in vitro.

Recently, we reported the therapeutic potential of mesenchymal stem/stromal cells (MSCs) from the maternal decidua basalis tissue of human term placenta (DBMSCs) to treat inflammatory diseases, such as atherosclerosis and cancer. DMSCs protect endothelial cell functions from the negative effects of oxidative stress mediators including hydrogen peroxide (H2 O2 ) and monocytes. In addition, DBMSCs induce the generation of anti-cancer immune cells known as M1 macrophages. Diabetes is another inflammatory disease where endothelial cells are injured by H2 O2 produced by high level of glucose (hyperglycaemia), which is associated with development of thrombosis. Here, we investigated the ability of DBMSCs to reverse the damaging effects of high levels of glucose on endothelial cells. DBMSCs and endothelial cells were isolated from human placental and umbilical cord tissues, respectively. Endothelial cells were incubated with glucose in presence of DBMSCs, and their functions were evaluated. The effect of DBMSCs on glucose- treated endothelial cell expression of genes was also determined. DBMSCs reversed the effects of glucose on endothelial cell functions including proliferation, migration, angiogenesis and permeability. In addition, DBMSCs modified the expression of several genes mediating essential endothelial cell functions including survival, apoptosis, permeability and angiogenesis. We report the first evidence that DBMSCs protect the functions of endothelial cells from the damaging effects of glucose. Based on these results, we establish that DBMSCs are promising therapeutic agents to repair glucose-induced endothelial cell injury in diabetes. However, these finding must be investigated further to determine the pathways underlying the protective role of DBMSCs on glucose-stimulated endothelial cell Injury.

Correction to: Preconditioning of Human Decidua Basalis Mesenchymal Stem/Stromal Cells with Glucose Increased Their Engraftment and Anti-diabetic Properties.

The author would like to correct the names for the below co-authors in the online published article.

Decidua Basalis Mesenchymal Stem Cells Favor Inflammatory M1 Macrophage Differentiation In Vitro.

Placental mesenchymal stem cells from maternal decidua basalis tissue (DBMSCs) are promising cells for tissue repair because of their multilineage differentiation and ability to protect endothelial cells from injury. Here, we examined DBMSC interaction with macrophages and whether this interaction could modulate the characteristics and functions of these macrophages. We induced monocytes to differentiate into M1-like macrophages in the presence of DBMSCs. DBMSC effects on differentiation were evaluated using microscopy, flow cytometry, and ELISA. DBMSC effects on M1-like macrophage induction of T cell function were also examined. The culture of DBMSCs with monocytes did not inhibit monocyte differentiation into M1-like inflammatory macrophages. This was confirmed by the morphological appearance of M1-like macrophages, increased expression of inflammatory molecules, and reduced expression of anti-inflammatory molecules. In addition, DBMSCs did not interfere with M1-like macrophage phagocytic activity; rather, they induced stimulatory effects of M1-like macrophages on CD4⁺ T cell proliferation and subsequent secretion of inflammatory molecules by T cells. We showed that DBMSCs enhanced the differentiation of M1-like inflammatory macrophages, which function as antitumor cells. Therefore, our findings suggest that DBMSCs are inflammatory cells that could be useful in cancer treatment via the enhancement of M1- like macrophages.

Isolation and Characterization of Mesenchymal Stem/Stromal Cells Derived from Human Third Trimester Placental Chorionic Villi and Decidua Basalis.

The decidua basalis and placental chorionic villi are critical components of maternal-fetal interface, which plays a critical role in normal placental development. Failure to form a proper maternal-fetal interface is associated with clinically important placental pathologies including preeclampsia and fetal growth restriction. Placental trophoblast cells are well known for their critical roles in establishing the maternal-fetal interface; however accumulating evidence also implicates mesenchymal stem/stromal cells that envelop the maternal and fetal blood vessels as playing an important role in the formation and efficient functioning of the interface. Moreover, recent studies associate abnormal mesenchymal stem/stromal cell function in the development of preeclampsia. Further research is needed to fully understand the role that these cells play in this clinically important placental pathology.The intimate relationship between maternal and fetal tissues at the interface poses significant problems in the enrichment of decidua basalis and chorionic villous mesenchymal stem/stromal cells without significant cross-contamination. The protocols described below for the enrichment and characterization of mesenchymal stem/stromal cells from the maternal-fetal interface produce highly enriched cells that conform to international standards and show minimal cross-contamination.

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by inflammation triggered by monocyte adhesion and increased endothelial cell proliferation. These events are manifest in inflammatory diseases, such as atherosclerosis. Therefore, our results suggest that DBMSCs could be usefully employed as a therapeutic strategy for atherosclerosis.

Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists.

High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate "universal" marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage.

Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta.

INTRODUCTION: Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation. METHODS: Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC. RESULTS: Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC. DISCUSSION: The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC.

Phenotypic and Functional Characterization of Mesenchymal Stem/Multipotent Stromal Cells From Decidua Parietalis of Human Term Placenta.

Mesenchymal stem/multipotent stromal cells (MSCs) from the human placenta show stem cell-like properties useful for regenerative medicine. Previously, we reported that MSCs isolated from the fetal part of human term placentae have characteristics, which make them a potential candidate for regenerative medicine. In this study, we characterized MSC isolated from the maternal part of human term placenta. The MSCs were isolated from the decidua parietalis (DPMSCs) of human placenta using a digestion method and characterized by colony-forming unit assay and the expression of MSC markers by flow cytometry technique. In addition, DPMSC differentiation into the 3 mesenchymal lineages was also performed. Moreover, the gene and protein expression profiles of DPMSCs were identified by real-time polymerase chain reaction and flow cytometry techniques, respectively. Furthermore, proteins secreted by DPMSCs were detected by sandwich enzyme-linked immunosorbent assays. Finally, the proliferation and migration potentials of DPMSCs were also determined. The DPMSCs were positive for MSC markers and negative for hematopoietic and endothelial markers, as well as costimulatory molecules and HLA-DR. Functionally, DPMSCs formed colonies and differentiated into chondrocytes, osteocytes, and adipocytes. In addition, they proliferated and migrated in response to different stimuli. Finally, they expressed and secreted many biological and immunological factors with multiple functions. Here, we carry out an extensive characterization of DPMSCs of human placenta. We report that these cells express and secrete a wide range of molecules with multiple functions, and therefore, we suggest that these cells could be an attractive candidate for cell-based therapy.

Mesenchymal Stem/Stromal Cells Derived From a Reproductive Tissue Niche Under Oxidative Stress Have High Aldehyde Dehydrogenase Activity.

The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother's tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDH(br)) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDH(br) cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDH(br) cells than CMSC. Finally, flow sorted ALDH(br) cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments.

Ectopic Bone Formation by Mesenchymal Stem Cells Derived from Human Term Placenta and the Decidua.

Mesenchymal stem cells (MSCs) are one of the most attractive cell types for cell-based bone tissue repair applications. Fetal-derived MSCs and maternal-derived MSCs have been isolated from chorionic villi of human term placenta and the decidua basalis attached to the placenta following delivery, respectively. Chorionic-derived MSCs (CMSCs) and decidua-derived MSCs (DMSCs) generated in this study met the MSCs criteria set by International Society of Cellular Therapy. These criteria include: (i) adherence to plastic; (ii) >90% expression of CD73, CD105, CD90, CD146, CD44 and CD166 combined with <5% expression of CD45, CD19 and HLA-DR; and (iii) ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. In vivo subcutaneous implantation into SCID mice showed that both bromo-deoxyuridine (BrdU)-labelled CMSCs and DMSCs when implanted together with hydroxyapatite/tricalcium phosphate particles were capable of forming ectopic bone at 8-weeks post-transplantation. Histological assessment showed expression of bone markers, osteopontin (OPN), osteocalcin (OCN), biglycan (BGN), bone sialoprotein (BSP), and also a marker of vasculature, alpha-smooth muscle actin (α-SMA). This study provides evidence to support CMSCs and DMSCs as cellular candidates with potent bone forming capacity.

Stem cell research and regenerative medicine at King Abdullah International Medical Research Center.

Translation of stem cell research from bench to bedside opens up exciting new therapeutic options for patients. Although stem cell research has progressed rapidly, its clinical applications have not kept pace. We report on the establishment of a stem cell research and regenerative medicine program at King Abdullah International Medical Research Center (KAIMRC). The purpose of this unit is to coordinate advanced stem cell research and translational outcomes with the goal of treating chronic human diseases, such as cancer, diabetes, cardiovascular, neurological, immunological, and liver diseases. Our first step in achieving this goal was to integrate the stem cells and regenerative medicine unit with our umbilical cord blood bank and bone marrow registry. This organizational structure will provide different sources for stem cells for research and clinical purposes, and facilitate our stem cell research and stem cell transplantation program. We are at an early and exciting stage in our program, but we believe that our progress to the international stage will be rapid and have a significant impact.

Analysis of homeobox gene action may reveal novel angiogenic pathways in normal placental vasculature and in clinical pregnancy disorders associated with abnormal placental angiogenesis.

Homeobox genes are essential for both the development of the blood and lymphatic vascular systems, as well as for their maintenance in the adult. Homeobox genes comprise an important family of transcription factors, which are characterized by a well conserved DNA binding motif; the homeodomain. The specificity of the homeodomain allows the transcription factor to bind to the promoter regions of batteries of target genes and thereby regulates their expression. Target genes identified for homeodomain proteins have been shown to control fundamental cell processes such as proliferation, differentiation, and apoptosis. We and others have reported that homeobox genes are expressed in the placental vasculature, but our knowledge of their downstream target genes is limited. This review highlights the importance of studying the cellular and molecular mechanisms by which homeobox genes and their downstream targets may regulate important vascular cellular processes such as proliferation, migration, and endothelial tube formation, which are essential for placental vasculogenesis and angiogenesis. A better understanding of the molecular targets of homeobox genes may lead to new therapies for aberrant angiogenesis associated with clinically important pregnancy pathologies, including fetal growth restriction and preeclampsia.

A novel combination of homeobox genes is expressed in mesenchymal chorionic stem/stromal cells in first trimester and term pregnancies.

Human chorionic mesenchymal stem/stromal cells (CMSCs) derived from the placenta are similar to adult tissue-derived MSCs. The aim of this study was to investigate the role of these cells in normal placental development. Transcription factors, particularly members of the homeobox gene family, play crucial roles in maintaining stem cell proliferation and lineage specification in embryonic tissues. In adult tissues and organs, stem cells proliferate at low levels in their niche until they receive cues from the microenvironment to differentiate. The homeobox genes that are expressed in the CMSC niche in placental tissues have not been identified. We used the novel strategy of laser capture microdissection to isolate the stromal component of first trimester villi and excluded the cytotrophoblast and syncytiotrophoblast layers that comprise the outer layer of the chorionic villi. Microarray analysis was then used to screen for homeobox genes in the microdissected tissue. Candidate homeobox genes were selected for further RNA analysis. Immunohistochemistry of candidate genes in first trimester placental villous stromal tissue revealed homeobox genes Meis1, myeloid ectropic viral integration site 1 homolog 2 (MEIS2), H2.0-like Drosophila (HLX), transforming growth factor ß-induced factor (TGIF), and distal-less homeobox 5 (DLX5) were expressed in the vascular niche where CMSCs have been shown to reside. Expression of MEIS2, HLX, TGIF, and DLX5 was also detected in scattered stromal cells. Real-time polymerase chain reaction and immunocytochemistry verified expression of MEIS2, HLX, TGIF, and DLX5 homeobox genes in first trimester and term CMSCs. These data suggest a combination of regulatory homeobox genes is expressed in CMSCs from early placental development to term, which may be required for stem cell proliferation and differentiation.

Downstream targets of the homeobox gene DLX3 are differentially expressed in the placentae of pregnancies affected by human idiopathic fetal growth restriction.

Human idiopathic fetal growth restriction (FGR) is associated with placental insufficiency. Previously, we reported that the expression of homeobox gene Distal-less 3 (DLX3) is increased in idiopathic FGR placentae and is a regulator of villous trophoblast differentiation. Here, we identify the downstream targets of DLX3 in trophoblast-derived cell lines. We modelled the high levels of DLX3 in FGR using an over-expression plasmid construct and complemented this using short-interference RNA (siRNA) for inactivation in cultured cells. Using a real-time PCR-based gene profiling, candidate target genes of DLX3 over-expression and inactivation were identified as regulators of trophoblast differentiation; GATA2 and PPARγ. The expression of GATA2 and PPARγ were further assessed in placental tissues and showed increased mRNA and protein levels in FGR-affected tissues compared with gestation-matched controls. We conclude that DLX3 orchestrates the expression of multiple regulators of trophoblast differentiation and that expression of these regulatory genes is abnormal in FGR.

Homeobox gene transforming growth factor ß-induced factor-1 (TGIF-1) is a regulator of villous trophoblast differentiation and its expression is increased in human idiopathic fetal growth restriction.

Abnormal trophoblast function is associated with human fetal growth restriction (FGR). Targeted disruption of homeobox gene transforming growth ß-induced factor (TGIF-1) results in placental dysfunction in the mouse. The role of human TGIF-1 in placental cell function is unknown. The aims of this study were to determine the expression of TGIF-1 in human idiopathic FGR-affected placentae compared with gestation-matched controls (GMC), to elucidate the functional role of TGIF-1 in trophoblasts and to identify its downstream targets. Real-time PCR and immunoblotting revealed that TGIF-1 mRNA and protein expression was significantly increased in FGR-affected placentae compared with GMC (n = 25 in each group P < 0.05). Immunoreactive TGIF-1 was localized to the villous cytotrophoblasts, syncytiotrophoblast, microvascular endothelial cells and in scattered stromal cells in both FGR and GMC. TGIF-1 inactivation in BeWo cells using two independent siRNA resulted in significantly decreased mRNA and protein of trophoblast differentiation markers, human chorionic gonadotrophin (CGB/hCG), syncytin and 3ß-hydroxysteroid dehydrogenase/3ß-honest significant difference expression. Our data demonstrate that homeobox gene TGIF-1 is a potential up-stream regulator of trophoblast differentiation and the altered TGIF-1 expression may contribute to aberrant villous trophoblast differentiation in FGR.

The immunomodulatory and neuroprotective effects of mesenchymal stem cells (MSCs) in experimental autoimmune encephalomyelitis (EAE): a model of multiple sclerosis (MS).

Mesenchymal stem cells (MSCs) are multipotent cells that differentiate into the mesenchymal lineages of adipocytes, osteocytes and chondrocytes. MSCs can also transdifferentiate and thereby cross lineage barriers, differentiating for example into neurons under certain experimental conditions. MSCs have anti-proliferative, anti-inflammatory and anti-apoptotic effects on neurons. Therefore, MSCs were tested in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), for their effectiveness in modulating the pathogenic process in EAE to develop effective therapies for MS. The data in the literature have shown that MSCs can inhibit the functions of autoreactive T cells in EAE and that this immunomodulation can be neuroprotective. In addition, MSCs can rescue neural cells via a mechanism that is mediated by soluble factors, which provide a suitable environment for neuron regeneration, remyelination and cerebral blood flow improvement. In this review, we discuss the effectiveness of MSCs in modulating the immunopathogenic process and in providing neuroprotection in EAE.

Immunosuppressive properties of mesenchymal stem cells.

Mesenchymal stem cells (MSC) can be isolated from different adult tissues including bone marrow, adipose tissue, cord blood and placenta. MSCs modulate the immune function of the major immune cell populations involved in alloantigen recognition and elimination, including antigen presenting cells, T cells, B cells and natural killer cells. Many clinical trials are currently underway that employ MSCs to treat human immunological diseases. However, the molecular mechanism that mediates the immunosuppressive effect of MSCs is still unclear and the safety of using MSC in patient needs further confirmation. Here, we review the cytokines that activate MSCs and the soluble factors produced by MSCs, which allow them to exert their immunosuppressive effects. We review the mechanism responsible, at least in part, for the immune suppressive effects of MSCs and highlight areas of research required for a better understanding of MSC immune modulation.