Utilizing epigenetic regulators to improve HSC-based lentiviral gene therapy.

ABSTRACT: The curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven in various diseases. However, the low number of true HSCs that can be collected from patients and the subsequent in vitro maintenance and expansion of true HSCs for genetic correction remains challenging. Addressing this issue, we here focused on optimizing culture conditions to improve ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explored the use of epigenetic regulators to enhance the effectiveness of HSC-based lentiviral (LV) gene therapy. The histone deacetylase inhibitor quisinostat and bromodomain inhibitor CPI203 each promoted ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single-cell RNA sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, whereas the addition of quisinostat or CPI203 improved the expansion of HSCs in transduction protocols. Notably, we demonstrated that the addition of quisinostat improved the LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effects of epigenetic regulators in HSC biology and their clinical applications to advance HSC-based gene correction.

Tregs from human blood differentiate into nonlymphoid tissue-resident effector cells upon TNFR2 costimulation.

Tregs can facilitate transplant tolerance and attenuate autoimmune and inflammatory diseases. Therefore, it is clinically relevant to stimulate Treg expansion and function in vivo and to create therapeutic Treg products in vitro. We report that TNF receptor 2 (TNFR2) is a unique costimulus for naive, thymus-derived Tregs (tTregs) from human blood that promotes their differentiation into nonlymphoid tissue-resident (NLT-resident) effector Tregs, without Th-like polarization. In contrast, CD28 costimulation maintains a lymphoid tissue-resident (LT-resident) Treg phenotype. We base this conclusion on transcriptome and proteome analysis of TNFR2- and CD28-costimulated CD4+ tTregs and conventional T cells (Tconvs), followed by bioinformatic comparison with published transcriptomic Treg signatures from NLT and LT in health and disease, including autoimmunity and cancer. These analyses illuminate that TNFR2 costimulation promoted tTreg capacity for survival, migration, immunosuppression, and tissue regeneration. Functional studies confirmed improved migratory ability of TNFR2-costimulated tTregs. Flow cytometry validated the presence of the TNFR2-driven tTreg signature in effector/memory Tregs from the human placenta, as opposed to blood. Thus, TNFR2 can be exploited as a driver of NLT-resident tTreg differentiation for adoptive cell therapy or antibody-based immunomodulation in human disease.

The Mac Is Back: The Role of Macrophages in Human Healthy and Complicated Pregnancies.

Pregnancy is a fascinating immunological paradox: the semi-allogeneic fetus generally grows without any complications. In the placenta, fetal trophoblast cells come into contact with maternal immune cells. Inaccurate or inadequate adaptations of the maternal immune system could lead to problems with the functioning of the placenta. Macrophages are important for tissue homeostasis, cleanup, and the repair of damaged tissues. This is crucial for a rapidly developing organ such as the placenta. The consensus on macrophages at the maternal-fetal interface in pregnancy is that a major proportion have an anti-inflammatory, M2-like phenotype, that expresses scavenger receptors and is involved in tissue remodeling and the dampening of the immune reactions. Recent multidimensional analyses have contributed to a more detailed outlook on macrophages. The new view is that this lineage represents a highly diverse phenotype and is more prevalent than previously thought. Spatial-temporal in situ analyses during gestation have identified unique interactions of macrophages both with trophoblasts and with T cells at different trimesters of pregnancy. Here, we elaborate on the role of macrophages during early human pregnancy and at later gestation. Their possible effect is reviewed in the context of HLA incompatibility between mother and fetus, first in naturally conceived pregnancies, but foremost in pregnancies after oocyte donation. The potential functional consequences of macrophages for pregnancy-related immune reactions and the outcome in patients with recurrent pregnancy loss are also discussed.

ESHRE guideline: recurrent pregnancy loss: an update in 2022.

STUDY QUESTION: What are the updates for the recommended management of women with recurrent pregnancy loss (RPL) based on the best available evidence in the literature from 2017 to 2022? SUMMARY ANSWER: The guideline development group (GDG) updated 11 existing recommendations on investigations and treatments for RPL, and how care should be organized, and added one new recommendation on adenomyosis investigation in women with RPL. WHAT IS KNOWN ALREADY: A previous ESHRE guideline on RPL was published in 2017 and needs to be updated. STUDY DESIGN SIZE DURATION: The guideline was developed and updated according to the structured methodology for development and update of ESHRE guidelines. The literature searches were updated, and assessments of relevant new evidence were performed. Relevant papers published between 31 March 2017 and 28 February 2022 and written in English were included. Cumulative live birth rate, live birth rate, and pregnancy loss rate (or miscarriage rate) were considered the critical outcomes. PARTICIPANTS/MATERIALS SETTING METHODS: Based on the collected evidence, recommendations were updated and discussed until consensus was reached within the GDG. A stakeholder review was organized after the updated draft was finalized. The final version was approved by the GDG and the ESHRE Executive Committee. MAIN RESULTS AND THE ROLE OF CHANCE: The new version of the guideline provides 39 recommendations on risk factors, prevention, and investigation in couples with RPL, and 38 recommendations on treatments. These includes 62 evidence-based recommendations-of which 33 were formulated as strong recommendations and 29 as conditional-and 15 good practice points. Of the evidence-based recommendations, 12 (19.4%) were supported by moderate-quality evidence. The remaining recommendations were supported by low (34 recommendations; 54.8%), or very low-quality evidence (16 recommendations; 25.8%). Owing to the lack of evidence-based investigations and treatments in RPL care, the guideline also clearly mentions those investigations and treatments that should not be used for couples with RPL. LIMITATIONS REASONS FOR CAUTION: The guidelines have been updated; however, several investigations and treatments currently offered to couples with RPL have not been well studied; for most of these investigations and treatments, a recommendation against using the intervention or treatment was formulated based on insufficient evidence. Future studies may require these recommendations to be revised. WIDER IMPLICATIONS OF THE FINDINGS: The guideline provides clinicians with clear advice on best practice in RPL, based on the best and most recent evidence available. In addition, a list of research recommendations is provided to stimulate further studies in RPL. Still, the absence of a unified definition of RPL is one of the most critical consequences of the limited scientific evidence in the field. STUDY FUNDING/COMPETING INTERESTS: The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, with the literature searches and with the dissemination of the guideline. The guideline group members did not receive payment.O.B.C. reports being a member of the executive board of the European Society for Reproductive Immunology and has received payment for honoraria for giving lectures about RPL in Australia in 2020. M.G. reports unconditional research and educational grant received by the Centre for Reproductive Medicine, Amsterdam UMC from Guerbet, Merck and Ferring, not related to the presented work. S.L. reports position funding from EXAMENLAB Ltd. and ownership interest by stock or partnership of EXAMENLAB Ltd (CEO). S.Q. reports being a deputy director of Tommy's National centre for miscarriage research, with payment received by the institution for research, staff time, and consumables for research. H.S.N. reports grants with payment to institution from Freya Biosciences ApS, Ferring Pharmaceuticals, BioInnovation Institute, the Danish ministry of Education, Novo Nordic Foundation, Augustinus Fonden, Oda og Hans Svenningsens Fond, Demant Fonden, Ole Kirks Fond, and Independent Research Fund Denmark and speakers' fees for lectures from Ferring Pharmaceuticals, Merck A/S, Astra Zeneca, IBSA Nordic and Cook Medical. She also reports to be an unpaid founder and chairman of a maternity foundation. M.-L.v.d.H. received small honoraria for lectures on RPL care. The other authors have no conflicts of interest to declare. DISCLAIMER: This guideline represents the views of ESHRE, which were achieved after careful consideration of the scientific evidence available at the time of preparation. In the absence of scientific evidence on certain aspects, a consensus between the relevant ESHRE stakeholders has been obtained.Adherence to these clinical practice guidelines does not guarantee a successful or specific outcome, nor does it establish a standard of care. Clinical practice guidelines do not replace the need for application of clinical judgment to each individual presentation, nor variations based on locality and facility type.ESHRE makes no warranty, express or implied, regarding the clinical practice guidelines and specifically excludes any warranties of merchantability and fitness for a particular use or purpose. (Full disclaimer available at www.eshre.eu/guidelines.).

IL3 Has a Detrimental Effect on Hematopoietic Stem Cell Self-Renewal in Transplantation Settings.

The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34+ from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34+ cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols.

Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions.

Introduction: Trophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells. Methods: After enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT. Results: The trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR. Conclusion: We verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

Toward more accurate prediction of future pregnancy outcome in couples with unexplained recurrent pregnancy loss: taking both partners into account.

OBJECTIVE: To identify, besides maternal age and the number of previous pregnancy losses, additional characteristics of couples with unexplained recurrent pregnancy loss (RPL) that improve the prediction of an ongoing pregnancy. DESIGN: Hospital-based cohort study in couples who visited specialized RPL units of two academic centers between 2012 and 2020. SETTING: Two academic centers in the Netherlands. PATIENTS: Clinical data from 526 couples with unexplained RPL were used in this study. INTERVENTION(S): None. MAIN OUTCOME MEASURES: The final model to estimate the chance of a subsequent ongoing pregnancy was determined using a backward selection process and internally validated using bootstrapping. Model performance was assessed in terms of calibration and discrimination (area under the receiver operating characteristic curve). RESULTS: Subsequent ongoing pregnancy was achieved in 345 of 526 couples (66%). The number of previous pregnancy losses, maternal age, paternal age, maternal body mass index, paternal body mass index, maternal smoking status, and previous in vitro fertilization/intracytoplasmic sperm injection treatment were predictive of the outcome. The optimism-corrected area under the receiver operating characteristic curve was 0.63 compared with 0.57 when using only the number of previous pregnancy losses and maternal age. CONCLUSIONS: The identification of additional predictors of a subsequent ongoing pregnancy after RPL, including male characteristics, is significant for both clinicians and couples with RPL. At the same time, we showed that the predictive ability of the current model is still limited and more research is warranted to develop a model that can be used in clinical practice.

Uncomplicated oocyte donation pregnancies display an elevated CD163-positive type 2 macrophage load in the decidua, which is associated with fetal-maternal HLA mismatches.

PROBLEM: The embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which may challenge the maternal immune system to tolerize the fetus. Decidual macrophages are essential in maintaining a healthy pregnancy, and type 2 macrophages may exhibit immune suppressive activity. We hypothesized that the composition of decidual macrophages is different between uncomplicated OD pregnancies and non-OD in vitro fertilization (IVF) pregnancies, and is related to fetal-maternal incompatibility. METHOD OF STUDY: Women with uncomplicated pregnancy were enrolled: 25 singleton OD pregnancies and 17 non-OD IVF pregnancies. The extent of immunohistochemical staining of CD14 (pan-macrophage marker) and CD163 (type 2 macrophage marker) in both decidua basalis and parietalis was quantitated by digital image analysis. Maternal and fetal DNA was typed for human leukocyte antigen (HLA)-A, -B, C, -DRB1, and -DQB1, and fetal-maternal HLA mismatches were calculated. RESULTS: OD pregnancies showed a higher percentage of CD163+ staining (P = .040) and higher CD163/CD14 ratio (P = .032) in the parietalis than non-OD IVF. The OD group was separated into a semi-allogeneic group (≤5 fetal maternal HLA mismatches) and a fully allogeneic group (> 5 mismatches). The HLA-fully-allogeneic OD group, but not the HLA-semi-allogeneic OD group, showed significantly elevated CD163/CD14 ratio in the parietalis compared with the non-OD IVF group (P < .05). CONCLUSIONS: Uncomplicated OD pregnancies display a higher CD163-positive cell fraction in the total decidual macrophage population compared to autologous pregnancies, which may suggest that a local type 2 macrophage-related mechanism is needed to compensate for the higher fetal-maternal HLA mismatch load.

Defining recurrent pregnancy loss: associated factors and prognosis in couples with two versus three or more pregnancy losses.

RESEARCH QUESTION: The definition of recurrent pregnancy loss (RPL) differs internationally. The European Society of Human Reproduction and Embryology (ESHRE) defines RPL as two or more pregnancy losses. Different definitions lead, however, to different approaches to care for couples with RPL. This study aimed to determine whether the distribution of RPL-associated factors was different in couples with two versus three or more pregnancy losses. If a similar distribution were found, couples with two pregnancy losses should be eligible for the same care pathway as couples with three pregnancy losses. DESIGN: This single-centre, retrospective cohort study investigated 383 couples included from 2012 to 2016 at the Leiden University Medical Center RPL clinic. Details on age, body mass index, smoking status, number of pregnancy losses, mean time to pregnancy loss and performed investigations were collected. The prevalence of uterine anomalies, antiphospholipid syndrome, hereditary thrombophilia, hyperhomocysteinaemia, chromosomal abnormalities and positive thyroid peroxidase antibodies were compared in couples with two versus three or more pregnancy losses. RESULTS: No associated factor was found in 71.5% of couples with RPL. This did not differ statistically between couples with two versus three or more pregnancy losses (73.6% versus 70.6%; P = 0.569). The distribution of investigated causes did not differ between the two groups. CONCLUSIONS: As the distribution of associated factors in couples with two versus three or more pregnancy losses is equal, couples with two pregnancy losses should be eligible for the same care pathway as couples with three. This study supports ESHRE's suggestion of including two pregnancy losses in the definition of RPL.

Mutation accumulation and developmental lineages in normal and Down syndrome human fetal haematopoiesis.

Children show a higher incidence of leukemia compared to young adolescents, yet their cells have less age-related (oncogenic) somatic mutations. Newborns with Down syndrome have an even higher risk of developing leukemia, which is thought to be driven by mutations that accumulate during fetal development. To characterize mutation accumulation in individual stem and progenitor cells of Down syndrome and karyotypically normal fetuses, we clonally expanded single cells and performed whole-genome sequencing. We found a higher mutation rate in haematopoietic stem and progenitor cells during fetal development compared to the post-infant rate. In fetal trisomy 21 cells the number of somatic mutations is even further increased, which was already apparent during the first cell divisions of embryogenesis before gastrulation. The number and types of mutations in fetal trisomy 21 haematopoietic stem and progenitor cells were similar to those in Down syndrome-associated myeloid preleukemia and could be attributed to mutational processes that were active during normal fetal haematopoiesis. Finally, we found that the contribution of early embryonic cells to human fetal tissues can vary considerably between individuals. The increased mutation rates found in this study, may contribute to the increased risk of leukemia early during life and the higher incidence of leukemia in Down syndrome.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes.

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen's flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

[Effectiveness of progesterone in pregnancy complications]. / Effect van progesteron bij zwangerschapscomplicaties.

This commentary reflects on the effectiveness of progesterone in early pregnancy complications. Several studies have investigated the role of progesterone in women with bleeding in early pregnancy and in women with recurrent miscarriages. These publications suggests that first trimester use of progesterone is not effective to prevent miscarriages when there is blood loss in the first trimester, and that it is also not effective to prevent new miscarriages in women with recurrent miscarriages in their history. However, subgroup analysis tells us it might be effective to prevent new miscarriages in women with blood loss in the first trimester and three earlier miscarriages. Expert groups in the Netherlands are currently considering recommendations on the prescription of progesterone for women with early pregnancy complications.

Loss of placental thrombomodulin in oocyte donation pregnancies.

OBJECTIVE: To investigate whether thrombomodulin dysregulation is involved in the development of preeclampsia after oocyte donation (OD). Women who become pregnant after OD are prone to develop preeclampsia, a syndrome characterized by an aberrant immunologic response, hypercoagulability, and endothelial dysfunction. A mediator of inflammation and coagulation is thrombomodulin, which has a possible role to play in this syndrome. DESIGN: Case-control study. SETTING: Not applicable. PATIENT(S): Placentas from 82 women with an uncomplicated pregnancy (48 naturally conceived, 21 IVF, and 33 OD pregnancies) and 9 women with an OD pregnancy complicated by preeclampsia have been studied. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Abundances of thrombomodulin protein and vitamin D receptor (VDR) were determined using immunohistochemistry; mRNA expression was determined using quantitative polymerase chain reaction. RESULT(S): Placental thrombomodulin protein abundance was lower in OD pregnancies (diffuse pattern in 45%) than in controls (diffuse pattern in 96%). Placental thrombomodulin mRNA expression was lower in OD pregnancies complicated by preeclampsia (0.72 ± 0.47) compared with in uncomplicated OD pregnancies (0.43 ± 0.18). Thrombomodulin expression correlated with inflammation and coagulation. VDR expression was decreased in OD pregnancies complicated by preeclampsia and was correlated with thrombomodulin mRNA. CONCLUSION(S): Pregnancies conceived through OD lose placental thrombomodulin expression. This loss is associated with an increased coagulation and inflammation and indicates that endothelial protection is diminished in OD pregnancies, which might be an explanation for the increased risk for preeclampsia. Vitamin D metabolism is dysregulated in OD pregnancies and might be a target for therapy.

Changes in cytokine production and composition of peripheral blood leukocytes during pregnancy are not associated with a difference in the proliferative immune response to the fetus.

We analyzed peripheral blood from women at term pregnancy for leukocyte composition, in vitro proliferative responses and cytokine production after nonspecific and fetus-specific stimulation. Maternal peripheral blood mononuclear cells (PBMCs) were collected and stimulated with umbilical cord blood (UCB) of the mother's own child, third-party UCB, nonspecific stimulus phytohemagglutinin, and anti-CD3 antibody, with PBMCs of nonpregnant women (cPBMC) as controls. Nine combinations of patient, child, third party child, and controls were selected on basis of sharing one human leukocyte antigen (HLA)-DR antigen. The response of mPBMC upon specific stimulation with fetal antigens was similar to that of cPBMC. No differences were found when comparing the mother's response upon stimulation to her own child with stimulation to that with a control child. Nonspecific stimulation with phytohemagglutinin and anti-CD3 antibody did not reveal a difference in proliferation rate between mPBMC and cPBMC. However, mPBMC contained a higher percentage of CD14(+) cells (p = 0.001) and activated T cells (CD25(dim), p < 0.0001), but a lower percentage CD16(-)CD56(bright) natural killer (NK) cells (p = 0.001) and CD16(+)CD56(+) NK cells (p = 0.003). mPBMC produced more interleukin (IL)-6, IL-10, and IL-17 compared with cPBMC (p < 0.05). We found differences in lymphocyte composition and cytokine production between mPBMC and cPBMC. These differences did not result in quantitative changes in proliferative responses during pregnancy compared with responses in nonpregnant controls.

Differential distribution and phenotype of decidual macrophages in preeclamptic versus control pregnancies.

Maternal immune tolerance of the semiallogeneic fetus is a complex phenomenon. Macrophages are an abundant cell population in the human decidua, and changes in distribution or phenotype may be involved in the development of preeclampsia. The aim of this study was to assess the distribution and phenotype of macrophages in preterm preeclamptic, preterm control, and term control placentas. Placentas of preterm preeclamptic (n = 6), preterm control (n = 5), and term control pregnancies (n = 6) were sequentially immunohistochemically stained for CD14, CD163, DC SIGN, and IL-10. The distributions of CD14(+), CD163(+), DC SIGN(+), IL-10(+), CD163(+)/CD14(+), DC SIGN(+)/CD14(+), and Flt-1/CD14(+) cells were determined by double staining and by digital image analysis of sequential photomicrographs. CD14 and CD163 expression increased significantly in preterm preeclamptic decidua basalis compared with preterm control pregnancies (P = 0.0006 and P = 0.034, respectively). IL-10 expression was significantly lower in the decidua parietalis of preterm preeclamptic pregnancies compared with preterm control pregnancies (P = 0.03). The CD163/CD14 ratio was significantly lower in the decidua basalis (P = 0.0293) and the DC SIGN/CD14 ratio was significantly higher in the decidua basalis (P < 0.0001) and parietalis (P < 0.0001) of preterm preeclamptic pregnancies compared with preterm control pregnancies. CD14(+) macrophages did express Flt-1. Alterations in distribution and phenotype of macrophages in the decidua of preterm preeclamptic pregnancies compared with control pregnancies may contribute to the pathogenesis of preeclampsia.