Are Microchimeric Fetal Stem Cells that Cross ...

Incognito: Are Microchimeric Fetal Stem Cells that Cross Placental Barrier Real Emissaries of Peace? Cosmin Andrei Cismaru, Laura Pop & Ioana Berindan-Neagoe

Stem Cell Reviews and Reports ISSN 1550-8943 Stem Cell Rev and Rep DOI 10.1007/s12015-018-9834-9

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Author's personal copy Stem Cell Reviews and Reports https://doi.org/10.1007/s12015-018-9834-9

Incognito: Are Microchimeric Fetal Stem Cells that Cross Placental Barrier Real Emissaries of Peace? Cosmin Andrei Cismaru 1

&

Laura Pop 1 & Ioana Berindan-Neagoe 1

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Chimerism occurs naturaly throughout gestation and can also occur as a consequence of transfusion and transplantation therapy. It consists of the acquisition and long-term persistence of a genetically distinct population of allogenic cells inside another organism. Previous reports have suggested that feto-maternal microchimerism could exert a beneficial effect on the treatment of hematological and solid tumors in patients treated by PBSCT. In this review we report the mechanism of transplacental fetal stem cell trafficking during pregnancy and the effect of their long-term persistence on autoimmunity, GVHD, PBSCT, cancer and stem cell treatment. Keywords Chimerism . Rechimerism . Transplantation . Allograft . Autograft . PBSC . GVHD . GVT . NIMA . Exosomes . Pluripotent stem cells . VSELs

Introduction Microchimerism describes the integration of a genetically individual population of cells inside another organism. In humans, the phenomenon can have different origins but is mostly vertical - acquired throughout gestation, and horizontal – following medical procedures such as blood transfusions, organ transplantation or stem cell transplantation. Vertical microchimerism refers to transplacental trafficking of cells between the mother and its fetuses during pregnancy or in dizygotic fetuses sharing the same blood source mostly through anastomoses between blood vessels. Fetal-maternal microchimerism (FMC) allows the acquisition of fetal cells into the mother’s organism during pregnancy. A physiological transfer of trophoblastic cells, red blood cells and fetal lymphocytes occurs without triggering rejection and persists for about 30 days after normal delivery and induced or spontaneous abortion. A small portion of the transferred cells are pluripotent fetal stem cells which remain active in the mother’s organism throughout life [1]. In the past * Cosmin Andrei Cismaru [email protected] 1

Research Center for Functional Genomics, Biomedicine and Translational Medicine, BIuliu Hatieganu^ University of Medicine and Pharmacy, 23 Gh. Marinescu street, 400337 Cluj-Napoca, Romania

decades, researchers have tried to establish whether they are simple relics of pregnancy, participate in tissue repair or may have a deletary role.

Current Understanding of Transplacental Cell Trafficking Transplant rejection is a well-defined immunologic phenomenon. The peaceful feto-maternal coexistence during mammalian pregnancy has been regarded as Ba paradox of nature^. As opposed to the classical notion, the placenta is not a unidirectional barrier, being a selective bidirectional gate. The exchange varies in intensity throughout pregnancy and consists of passive and active transfer, an important mechanism of cell trafficking being represented by effracting the trophoblast [2]. Major histocompatibility complex acts in a particular manner in trophoblastic cells of the placenta, permitting a bidirectional transfer of substances, immunoglobulins and selected cells [3]. Pregnancy modulates the mother’s immune system to allow the development of the fetus. Fetal cells and circulating DNA fragments can be detected in maternal blood as early as the 6th week of gestation. Their number increases gradually to the 36th week and decreases progressively after parturition, suggesting a dynamic phenomenon [4, 5]. The number of microchimeric fetal cells in maternal blood increases by 100 times after pregnancy termination procedures and is even larger in surgical abortions

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compared to chemical terminations. Their number is also influenced by fetal or placental anomalies [6]. The placenta seems responsible for immunologic balance of pregnancy where rejection reaction is minimal and facilitation is intense. The maternal immune system can distinctively tolerate the engraftment of paternally derived tumor cells [7]. The placenta has a particular structural organization that permits fetal cells expressing paternal alloantigens to promote a friendly cohabitation with the maternal immune system [8]. Even though the exact cellular interactions and mechanisms involved in this type of maternal lymphocyte tolerance are not yet entirely known, some mechanisms of the immune reactions taking place at placental level are now beginning to be uncovered. These include suppression of NK cells and CD8 + T cells activation by HLA-G molecules expressed by fetal trophoblasts [9], lysis of activated maternal lymphocytes by FAS ligand expressing fetal trophoblasts, TNF related apoptosis-inducing ligand receptor, PD-L1 [10, 11], indoleamin 2.3 dioxigenase [12], expression of CRRY complement regulatory protein in placental structures [13], maternal CD4 + CD25+ regulatory T cells in the maternal preventing T cell alloreactivity [14, 15]. This last physiological mechanism strongly supports the hypothesis that amelioration of autoimmune diseases during pregnancy is due to the resulting enhancement of regulatory T cell function. Much of these cell to cell interactions and facilitation signals are being carried out throughout plasma membranederived shed microvesicles and endosomally secreted exosomes of syncytiotrophoblast. Three roles have been attributed to exosome interactions during pregnancy: (I) Impairment of T-cell immune response by down-regulation of the intracellular signaling through the CD3-z chain of the accessory molecule CD3 and the enzyme JAK3 in the placental exosomes. (II) Down-regulation of de major activating NK cell receptor NKG2D on cytotoxic T-, NK- and γδT cells by placental exosomes bearing NKG2D ligand with a consequent impairment of the maternal cytotoxicity and protection of the fetal allograft against maternal cytotoxic immune attack. (III) An active functional form of the apoptosis-inducing molecules FasL, TRAIL and PD-L1 being carried in placental exosomes which are able to induce apoptosis in activated immune cells. These functions make placental exosomes important emissaries of peace in the establishment of the maternal tolerance towards the fetus [16].

FMC and the Natural Bariers of the Organism The placental barrier is not the only immunologic organ permitting microchimeric fetal stem cells (MFSC) a selective transfer to the maternal circulation and tissues. MFSC have been shown to cross the blood-brain barrier into the cerebrum of the mother, many decades after pregnancy, and adopt a neuronal phenotype [17]. Their passage through natural

barriers of the organism demonstrates facilitation signal expression and low immunogenicity. To evaluate fetal microchimerism in human adult tissues, whole cells can be detected using in situ hibridisation (FISH), and DNA originating from the fetus can be amplified using polymerase chain reaction (PCR). Examination of gender mismatches based on the presence of Y chromosome genes in maternal tissues after pregnancies with male fetuses has been regarded as a practical technique as it only requires a sample from the mother. Authors also examined non shared HLA-DR alleles between mother and fetus to differentiate one genetic material from another in a similar manner as in evaluating chimerism after allogenic stem cell transplantation. This last technique requires analyzing samples from both mother and fetus [18]. Bidirectional passage of cells between mother and fetus during normal pregnancy can result in the presence of maternal cells in the fetal circulation (maternal microchimerism), the extent after parturition being evaluated at 42%, whereas fetal microchierism at 51% in normal pregnancies [19]. These maternal cells have been identified in healthy, immunocompetent individuals into adult life [18]. Similar to fetal microchimerism, maternal microchimerism has been associated with tissue repair in the fetus, although maternal microchimeric stem cells have been also shown to home to the site of autoimmune disease lesions [20, 21].

FMC and Tissue Repair Maternal blood and tissue harbor a variety of microchimeric fetal stem cell types, which have been identified by phenotyping. These comprise B or T lymphocytes, natural killer cells and CD34+ cells, monocytes and trophoblasts [22–25], cardiomyocytes [26], kidney tubular epithelium [27] hepatocytes [28] endothelial cells [29], thyrocytes [30] and also neurons and glia [31]. Differentiated fetal microchimeric stem cells (FMSCs) in maternal tissue have been shown to arise from mesenchimal stem cells [31] endothelial progenitor cells (EPC) [29] and lymphoid progenitors [24, 32]. The variety of phenotypes exhibited supports the stemness potential of FMSCs in the maternal structures in which they incorporate [33]. Mouse models show FMSCs originated from the epiblast or primitive endoderm of the embrio, that may have trafficked into maternal blood after implantation. This presumed state of migratory potential attributes them a level of pluripotency as embryonic stem cells derived from the inner mass. Throughout gestation, mothers can acquire fetal lymphoid progenitors that develop into functional T cells. This fetal cell microchimerism can have a direct effect on maternal health, as it was shown in the case of one immunocompromised murine female whose entire immune system was replaced by fetal immune cells [24]. Extensive fetal cell microchimerism was also shown in liver tissue of a pregnant woman with hepatitis C from


drug addiction and abuse. She obtained complete remission after liver phenotype MFSC replaced liver tissue despite her early discontinuance of the treatment. This led the authors to conclude that fetal cells may participate in mother’s tissue repair processes [34]. Isolation and characterization of fetal cells within a primitive CD34+ adherent stem cell population in maternal blood has identified MFSC with multitissue potential for differentiation, expressing pluripotent stem cell markers such as Oct4, Nanog and Rex1. Being part of a CD34+ adherent subpopulation of mixed maternal and fetal stem cells with embryonic differentiation capabilities, these isolated fetal microchimeric stem cells would have the potential to colonize multiple tissues and organs [35]. Early embryogenesis in the development of the fetus may deposit very small embryonic-like stem cells (VSELs) in certain developing organs during early gastrulation, which may persist into adulthood [36]. Existence of such VSELs niche in adult tissues may explain how MFSC which express embryonic markers, could home to such niche and coexist with maternal VSELs, allowing isolation of mixed populations of stem cells with embryonic differentiation potential. Existence of such niche would also explain how maternal VSELs could home to fetal niche during pregnancy generating maternal microchimerism that persists into adulthood. Supporting the idea of VSELs probably being involved in fetal-maternal microchimerism are some studies of the umbilical cord pluripotent stem cells [37].

FMC and Autoimmunity Immunologic implications of the fetal microchimerism imply that certain disease of the women considered until now auto-immune, could in reality be allo-immune, induced by the FMSCs, being responsable for a chronic GVHD-like reaction. Microchimerism and autoimmune disease association has been widely investigated as shown in Table 1. Some authors suggest that FMC may occur in a wide disease spectrum and can occur in both autoimmune and non-autoimmune forms of some pathologies. Fetal and maternal microchimerism is also found in healthy individuals without any manifestations of autoimmune diseases and in immunocompetent women with a history of an uncomplicated delivery [34]. These studies, taken together, suggest that FMC may be a relatively common occurrence in women with both autoimmune and non-autoimmune diseases and It is now believed that fetal stem cells do not trigger autoimmune diseases but are instead recruited at the affected site by maternal tissue when the lesion reaches a certain threshold [50]. This is supported by the results of studies that reported the absence of FMC in some lesions of autoimmune disease [51].

FMC and Graft Versus Host Disease (GVHD) In a wide spectrum of hematological malignancies, immunodeficiency syndromes, bone marrow failure and some congenital metabolic disorders, a curative procedure is represented by allogeneic hematopoietic stem cell transplantation (HSCT). The most important complication of allogeneic HSCT is GVHD. HLA incompatibility between the donor and recipient is the most important factor that dictates the severity of GVHD [52]. In patients who don’t have related donor which is HLAidentical, an alternative transplant substitute has been HSCT from a haplo-identical related donor. The convenience of haplo-identical HSCT is that most patients have a rapidly available donor within their family members. This doesn’t come without disadvantages as the consequence of crossing the MHC barrier may consist in graft rejection and GVHD. Determining tolerable HLA mismatches that could be less immunogenic to the transplanted patient in what concerns the GVHD would be of great comfort. Successful haploidentical, three loci mismatched HSCT has been reported by some authors based on FMC [53]. The transplant that originated from a FMC positive mother did not trigger GVHD. The fesability of the method was evaluated in several groups [54–56] and was soon found to be a viable alternative. Further evidence supporting these findings was reported in a study using International Bone Marrow Transplant Registry haplo identical HSCT for the patients. A reduced frequency of acute GVHD vas registered in patients given HSCT from a noninherited maternal HLA antigen mismatched sibling than that from a noninherited paternal antigen mismatched sibling donor, based on feto-maternal microchimerism [57]. Even after nonmyeloablative conditioning, successful engraftment was reported from an inherited paternal HLA antigen (IPA)/NIMAmismatched donor in patients with aplastic anemia, hematological malignancies and renal cell carcinoma [4, 58, 59]. The tolerogenic non inherited maternal antigen (NIMA) effect is a form of naturally acquired immunological hyporesponsiveness, which is believed to be a consequence of fetal or neonatal tolerance to NIMAs. An essential role in the induction of NIMA effects after allogeneic HSCT is attributed to CD4 + CD25+ regulatory T cells. Specific tolerance is believed to be the consequence of exposure to NIMAs which actively induces an immunological mechanism that supports manteinance of NIMAs [60].

FMC and Prediction of Therapeutic Response in PBSC Transplanted Patients Animal models indicate that while T-cell-depletion and mieloablative regimens in hematopoietic stem cell transplantation allow transplantation tolerance with the formation of full hematopoietic chimeras, in mixed chimeras, allogenic hematopoietic cells coexist with recipient cells following non-T-

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Association of fetal microchimerism and autoimmune disease

Dissease

Author and year of publication

Main findings

Systemic sclerosis

Artlett CM et al. [38] Johnson KL et al. [39] Ohtsuka T et al. [40] Lambert NC et al. [41] Ichikawa N et al. [42]

Presence of MFSC in lesions of the skin from parous women with systemic sclerosis (SSc) and in other organs in SSc patients but not controls Larger number of fetal cells in peripheral blood of women with scleroderma and in the skin from SSc patients than controls. Pontrols showed less fetal cells compared to SSc patients

Hashimoto disease Graves disease

Klintschar M et al. [43] Ando T et al. [44] Khosrotehrani et al. [24]

Increased frequency of male microchimerism in female thyroid Increased frequency of male microchimerism in female thyroid vs controls Male cells were almost as frequent in some other non-autoimmune thyroid pathologies

Primary biliary cirrhosis

Fanning PA et al. [45] Selva O’Callaghan A et al. [46]

Fetal cell microchimerism may be implicated in the pathogenesis of PBC Fetal microchimerism does not appear to have a major contribution in patients with PBC

Systemic Lupus

Kremer Hovinga IC et al. [47] Florim GM et al. [48]

Rheumatoid arthritis

Nelson JL et al. [49]

Chimerism occurs twice as often in lupus nephritis as in normal kidneys. Patients with positive kidney biopsy for fetal microchimerism had better kidney function than patients without microchimerism. Pregnancy ameliorates RA, the degree of amelioration being correlated with the frequency of fetal microchimeric stem cells

cell-depleted nonmyeloablative regimens. Mixed chimerism has several advantages over full chimerism in approaching induction of tolerance, one of them being a superior immunocompetence produced across MHC barriers [61].

In humans, the development of mixed chimerism in transplanted patients has been associated with transplant tolerance, both in organ and in bone marrow transplantation [62–64].

Fig. 1 Schematic representation of human placenta’s structure (adapted from 2012 Pearson Education Inc. and Georgiades et al.) and assumed mechanisms of cell trafficking from fetus to its mother. Speculated mechanisms of fetomaternal cell transfer include (a) deportation of

trophoblasts from the maternal vessels and intervillous space; (b) microtraumatic hemorrhage; and (c) cell adhesion and transmigration over the placental barrier


Fig. 2 Schematic representations of feto-placental and blood-brain barriers and proposed molecular mechanisms of cellular adhesion and transmigration. (I) The placental barrier includes fetal capillary endothelial cells (fcec), an endothelial basement membrane (ebm), the villous core (vc) which contains pericytes (p) and extracellular matrix, a trophoblastic basement membrane (tbm), in the first trimester a multinucleated syncytium of syncytiotrophoblasts (ss) and a layer of proliferative cytotrophoblasts (ct). It is proposed that fetal cells could adhere and transmigrate across the placental barrier similar to

lymphocytes trafficking the blood-brain barrier. (B) (as described by Engelhardt). Cells expressing a4b1 are grasped by VCAM-1 expressed by endothelial cells. There is a rapid activation stage (seconds) possibly involving lymphoid chemokines CCL21/SLC and CCL19/ELC. There is a prolonged adhesion stage (hours) followed by gradual transmigration (hours) conditioned upon binding of LFA-1 to ICAM-1 and/or ICAM-2 on the endothelial cells. It is theorized that a similar molecular mechanism may explain fetal cell trafficking across the blood-brain barrier and the placental barrier

Feto-maternal microchimerism may result in the induction of tolerance in bone marrow transplantation through exposure

to NIMA’s. Supporting this idea was a study showing the beneficial effect of fetal stem cell engraftment through PBSCT. This was associated with increased survival time and response rate and reduced risk of GVHD in patients with solid tumors treated by PBSCT obtained from feto-maternal microchimerism positive donors [65] (Figs. 1 and 2). The resulting mixed chimerism consisting of recipient cells, allogenic donor cells and allogenic fetal cells is a distinct type of mixed chimerism as it implies rechimerization of fetal stem cells from the mother as a donor, to the patient as a recipient. The rechimerism potential is a relevant ability of allogenic fetal stem cells as they have the capacity to chimerize into the mother through pregnancy and rechimerize into the patient through PBSCT. Rechimerism can occur both in a horizontal manner following transplant procedures and also in a vertical manner from an older sibling to the younger one through fetomaternal microchimerism during pregnancy. (Fig. 3 and Fig. 4). Evaluating the presence of feto-maternal microchimerism before PBSCT and evaluating the ability of IFN-γ spotforming cells against NIMA through MLR-ELISPOT assay might be a valuable tool in predicting the immunological reactivity of donor T cells against the recipient in NIMAmismatched or maternal haploidentical HSCT [66]. Alltogether, it could predict transplant tolerance, GVHd, therapeutic response rate and survival time in these patients.

Fig. 3 Simplified representation of the mechanism of vertical rechimerism. Fetal microchimeric stem cells are acquired throughout pregnancy from sibling 1 by feto-maternal microchimerism. Second pregnancy allows rechimerism of microhimeric fetal stem cells from sibling 1 to sibling 2

Author's personal copy Stem Cell Rev and Rep Fig. 4 Simplified representation of the mechanism of horizontal rechimerism. Fetal microchimeric stem cells are integrated in the mother’s organism through fetalmaternal microchimerism. Donor stem cells containing MFSC are being transplanted by allogeneic stem cell transplantation. There is a mixed chimerism consisting of donor derived stem cells and fetal microchimeric stem cells in the recipient organism. Fetal microchimeric stem cells engraft for a second time in a different organism generating rechimerism

FMC and Cancer A high proportion of male DNA has been reported in healthy women giving birth to male fetuses. Allogeneic inhabitance correlates with protection from breast cancer, but the result is dose dependent. Chimerism both above or below a certain threshold has been shown to be associated with cancer; Within these limits, however, at levels similar to endogenous stem/progenitor densities in the mammary gland, chimeric Y chromosome allogeneic cells were protective. The significance is of a probable niche competition mechanism in microchimerism stem cell biology [67]. Reports on FMC in peripheral blood and tissue from breast cancer patients, indicated by amplification on polymer chain reaction of fetal genes showed lower numbers of FMSCs in the circulation of women with breast cancer in contrast with healthy individuals [5, 68, 69]. A comparisons between patients with hematological and solid tumors versus healthy individuals indicated that FMSCs are recruited to the primary site of the disease since their numbers were increased in the blood of hematologic malignancies as compared to solid tumors [70]. The phenomenon was further highlighted through simultaneous examination of the presence of fetal cells both in blood and in tumor tissue, performed in a thyroid follow-

up study that tested female patients with papillary thyroid carcinoma and healthy controls. Although the number of FMSCs was lower in patients than in healthy controls, FMSCs were found at tumor site in patients, supporting the hypothesis that FMSCs are being recruited to the site of damaged tissue from maternal niche [71, 72]. Relationship between FMC and melanoma has also been evaluated. Fetal cells were found in 62% of the studied melanomas versus 12% of benign nevi. Chimeric cells adopted an endothelial phenotype that occasionally clustered and took part in the formation of vessels probably through stimuli by VEGF, including lymphatic vessels. As lymphangiogenesis is a bad prognosis factor as it favours metastatic spread, FMSCs were attributed a deletary role in malignant melanoma [73]. It’s been theorized that fetal cell microchimerism may protect against breast cancer by ensuring immune surveillance in the blood-flow. This is in accordance with the epidemiological studies that show a lower incidence of breast cancer in parous women. Authors attributed FMSCs a graft versus tumor effect (GVT).Based on the current understanding of the immunology of allogeneic bone marrow transplantation among family members, three potential mechanisms for the GVT effect of FMSCs have been proposed. They


include (a) the recognition by fetal T cells of allogeneic cancer antigens followed by a direct attack of the cancer cells; (b) compensation of the mothers ineffective antigen presenting cells’s activity upon maternal self-antigen, aiding to produce a proper immune response against cancer cells; and (c) exerting of a cytolitic activity by NK fetal cells, uninhibited by mismatched antigens on the cancer cells [74].

FMC and Stem Cell Therapy MFSC offer noticeable therapeutic potential in contrast to their somatic and fetal anexes counterparts in plasticity and over embryonic and induced pluripotent stem cells in oncogenic potential and ethics [75]. MFSC express immune facilitation signals and have longer telomeres than their adult counterparts which allow them to persist for many decades inside the recipient organism. Standing out for their potential in therapy is the expression of embryonic pluripotency markers such as Nanog, Oct4 and Rex1 [35], thus being able to differentiate into almost any cell in the body. Previous studies have shown that patients with metastatic solid tumors can benefit from activated allogenic haploidentical peripheral blood stem cell (haplo-PBSC) treatment as it exerts an anti-tumor effect (GVT). An enhancement of thetherapeutic effect of the haplo-PBSC has been previously reported on the contribution of feto-maternal microchimerism to the allogenic haplo PBSCT [65]. Supporting this research is a prospective phase 1/2 clinical trial in which patients with advanced-stage chemotherapy-refractory solid tumors have been treated with activated haploidentical peripheral blood stem cells (haplo-PBSC). The trial showed that both overall survival and progression free survival of fetal-maternal microchimerism positive patients was higher than in fetalmaternal microchimerism negative patients [76]. Developing new techniques of selection and enrichment of FMSCs from fetal-maternal microchimerism positive donors in order to benefit from their therapeutical effect in the treatment of cancer by allogenic haplo-PBSCT represents a very close perspective [77].

Clinical Utility of FMC Clinical utility of FMC has been validated in prediction of pregnancy complications [78] and prenatal testing for aneuploidies [79, 80]. Stem cell transplantation is a potentially curative therapy for several hematological and oncological malignancies where autologus and allogeneic transplantation are part of the therapeutic protocols. Theese include Hodgkin and non-Hodgkin lymphoma, multiple myeloma, acute and chronic leukemia, neuroblastoma and germ cell neoplasms where it is regarded as standard of care, and breast carcinoma, renal carcinoma,

and selected other malignancies where this approach is developmental [81]. The outcomes include replenishing the bone marrow niche with healthy stem cells in autologous grafts and benefiting the graft vs tumor effect in allogeneic grafts. Engraftment failure as well as acute and chronic GVHD are some of the most common and undesired complications of allografts whereas in autografts, GVHD is rarely seen but GVT effect is not characteristic. Several techniques of improving the engraftment and longterm outcome of stem cell transplantation are currently being used and some others are being tested. These include: a) using reduced intensity nonmieloablative conditioning to preserve bone marrow niche microenvironment and avoid procedurerelated toxicity and mortality; b) favoring bone marrow aspiration over peripheral blood cytapheresis in order to avoid mobilization by G-CSF in the donor and reduce frequency of chronic GVHD in the patient; c) using intra-bone catheters for the infusion of the stem cells in order to increase homing and reduce stuckage in capilary systems of organs; d) using regular infusions of donor lymphocytes to promote donor chimerism and GVT effect; The fifth technique could be selecting donors positive for fetal-maternal chimerism in order to benefit from the additional tolerogenic effect reducing GVHD and take advantage of the stemness potential of the younger MFSC. In the same manner, autografts in patients positive for fetal microchimerism could bennefit from the lack of GVHD but profit from the GVT effect of the MFSC. Although it is not yet clear whether MFSC have both beneficial and detrimental effects in certain pathologies, exerting a tolerogenic effect as emissaries of peace can potentially improve the outcome of current stem cell transplantation techniques where graft acceptance plays a crucial role.

Compliance with Ethical Standards Conflict of Interests All authors have read and approved this version of the article, and due care has been taken to ensure the integrity of the work. No part of this article has been published or submitted elsewhere. No financial conflict of interest exists in the submission of this manuscript.

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