Daniela Ulrich

Toward the use of endometrial and menstrual blood mesenchymal stem cells for cell-based therapies

Introduction: Bone marrow is a widely used source of mesenchymal stem cells (MSCs) for cell-based therapies. Recently, endometrium – the highly regenerative lining of the uterus – and menstrual blood have been identified as more accessible sources of MSCs. These uterine MSCs include two related cell types: endometrial MSCs (eMSCs) and endometrial regenerative cells (ERCs). Areas covered: The properties of eMSCs and ERCs and their application in preclinical in vitro and in vivo studies for pelvic organ prolapse, heart disorders and ischemic conditions are reviewed. Details of the first clinical Phase I and Phase II studies will be provided. Expert opinion: The authors report that eMSCs and ERCs are a readily available source of adult stem cells. Both eMSCs and ERCs fulfill the key MSC criteria and have been successfully used in preclinical models to treat various diseases. Data on clinical trials are sparse. More research is needed to determine the mechanism of action of eMSCs and ERCs in these regenerative medicine models and to determine the long-term benefits and any adverse effects after their administration.

Vascular Endothelial Expression of Indoleamine 2,3-Dioxygenase 1 Forms a Positive Gradient towards the Feto-Maternal Interface

We describe the distribution of indoleamine 2,3-dioxygenase 1 (IDO1) in vascular endothelium of human first-trimester and term placenta. Expression of IDO1 protein on the fetal side of the interface extended from almost exclusively sub-trophoblastic capillaries in first-trimester placenta to a nearly general presence on villous vascular endothelia at term, including also most bigger vessels such as villous arteries and veins of stem villi and vessels of the chorionic plate. Umbilical cord vessels were generally negative for IDO1 protein. In the fetal part of the placenta positivity for IDO1 was restricted to vascular endothelium, which did not co-express HLA-DR. This finding paralleled detectability of IDO1 mRNA in first trimester and term tissue and a high increase in the kynurenine to tryptophan ratio in chorionic villous tissue from first trimester to term placenta. Endothelial cells isolated from the chorionic plate of term placenta expressed IDO1 mRNA in contrast to endothelial cells originating from human umbilical vein, iliac vein or aorta. In first trimester decidua we found endothelium of arteries rather than veins expressing IDO1, which was complementory to expression of HLA-DR. An estimation of IDO activity on the basis of the ratio of kynurenine and tryptophan in blood taken from vessels of the chorionic plate of term placenta indicated far higher values than those found in the peripheral blood of adults. Thus, a gradient of vascular endothelial IDO1 expression is present at both sides of the feto-maternal interface.

A preclinical evaluation of alternative synthetic biomaterials for fascial defect repair using a rat abdominal hernia model.

Introduction Fascial defects are a common problem in the abdominal wall and in the vagina leading to hernia or pelvic organ prolapse that requires mesh enhancement to reduce operation failure. However, the long-term outcome of synthetic mesh surgery may be unsatisfactory due to post-surgical complications. We hypothesized that mesh fabricated from alternative synthetic polymers may evoke a different tissue response, and provide more appropriate mechanical properties for hernia repair. Our aim was to compare the in vivo biocompatibility of new synthetic meshes with a commercial mesh. Methods We have fabricated 3 new warp-knitted synthetic meshes from different polymers with different tensile properties polyetheretherketone (PEEK), polyamide (PA) and a composite, gelatin coated PA (PA+G). The rat abdominal hernia model was used to implant the meshes (25×35 mm, n = 24/ group). After 7, 30, 60, 90 days tissues were explanted for immunohistochemical assessment of foreign body reaction and tissue integration, using CD31, CD45, CD68, alpha-SMA antibodies. The images were analysed using an image analysis software program. Biomechanical properties were uniaxially evaluated using an Instron Tensile® Tester. Results This study showed that the new meshes induced complex differences in the type of foreign body reaction over the time course of implantation. The PA, and particularly the composite PA+G meshes, evoked a milder early inflammatory response, and macrophages were apparent throughout the time course. Our meshes led to better tissue integration and new collagen deposition, particularly with the PA+G meshes, as well as greater and sustained neovascularisation compared with the PP meshes. Conclusion PA, PA+G and PEEK appear to be well tolerated and are biocompatible, evoking an overlapping and different host tissue response with time that might convey mechanical variations in the healing tissue. These new meshes comprising different polymers may provide an alternative option for future treatment of fascial defects.

Influence of Reproductive Status on Tissue Composition and Biomechanical Properties of Ovine Vagina

Objective To undertake a comprehensive analysis of the biochemical tissue composition and passive biomechanical properties of ovine vagina and relate this to the histo-architecture at different reproductive stages as part of the establishment of a large preclinical animal model for evaluating regenerative medicine approaches for surgical treatment of pelvic organ prolapse. Methods Vaginal tissue was collected from virgin (n = 3), parous (n = 6) and pregnant sheep (n = 6; mean gestation; 132 d; term = 145 d). Tissue histology was analyzed using H+E and Massons Trichrome staining. Biochemical analysis of the extracellular matrix proteins used a hydroxyproline assay to quantify total collagen, SDS PAGE to measure collagen III/I+III ratios, dimethylmethylene blue to quantify glycosaminoglycans and amino acid analysis to quantify elastin. Uniaxial tensiometry was used to determine the Youngs modulus, maximum stress and strain, and permanent strain following cyclic loading. Results Vaginal tissue of virgin sheep had the lowest total collagen content and permanent strain. Parous tissue had the highest total collagen and lowest elastin content with concomitant high maximum stress. In contrast, pregnant sheep had the highest elastin and lowest collagen contents, and thickest smooth muscle layer, which was associated with low maximum stress and poor dimensional recovery following repetitive loading. Conclusion Pregnant ovine vagina was the most extensible, but the weakest tissue, whereas parous and virgin tissues were strong and elastic. Pregnancy had the greatest impact on tissue composition and biomechanical properties, compatible with significant tissue remodeling as demonstrated in other species. Biochemical changes in tissue protein composition coincide with these altered biomechanical properties.

Changes in haemodynamic and autonomous nervous system parameters measured non-invasively throughout normal pregnancy

OBJECTIVE To examine non-invasively haemodynamic and autonomous parameters throughout normal pregnancy. STUDY DESIGN We used the Task Force Monitor 3040i system to retrieve, record, and calculate haemodynamic as well as autonomous parameters. 20 healthy women were included and scheduled for longitudinal examinations throughout normal pregnancy. Heart rate (HR), blood pressure (BP), stroke volume (SV), cardiac output (CO), systemic vascular resistance (SVR), heart rate variability (HRV), blood pressure variability (BPV), and baroreceptor sensitivity (BRS) were measured. Measurements were performed at gestational week 10(+0)-13(+6), 15(+0)-18(+6), 20(+0)-22(+6), and >30(+0). RESULTS HR increased during gestation showing a significant increase at III versus I trimester (74 bpm vs. 88 bpm, P<.05). Mean arterial pressure remained stable until III trimester, when a significant increase compared to I trimester could be noted (78 mm Hg vs. 86 mm Hg, P<.05). SV and CO remained relatively stable in I and II trimester, and in III trimester significant decreases were observed. In contrast, SVR increased significantly at III trimester (P<.001). Whereas HRV and BPV did not change at different gestational ages, BRS was significantly lower in III trimester compared to I trimester values (P<.05). CONCLUSION The non-invasive determination of cardiovascular and autonomous parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular and autonomous changes in pathological conditions in pregnancy such as hypertensive disorders.

Use of a visual analog scale for evaluation of bother from pelvic organ prolapse.

A specified anatomical degree of prolapse may cause no bother in one individual and a severe form of bother in another. The aim of this study was to determine the factors contributing to the degree of bother due to a given degree of prolapse, to help with the clinical evaluation of patients and planning of preventive intervention trials and surgical treatment.

Mesenchymal stem/stromal cells in post-menopausal endometrium

STUDY QUESTION Does post-menopausal endometrium contain mesenchymal stem/stromal cells (MSC) that have adult stem cell properties and can be prospectively isolated from a biopsy? SUMMARY ANSWER Perivascular W5C5(+) cells isolated from post-menopausal endometrial biopsies displayed characteristic MSC properties of clonogenicity, multipotency and surface phenotype irrespective of whether the women were or were not pre-treated with estrogen to regenerate the endometrium. WHAT IS KNOWN ALREADY Recently MSCs have been identified in human premenopausal endometrium, and can be prospectively isolated using a single marker, W5C5/SUSD2. STUDY DESIGN, SIZE, DURATION Endometrial tissue of both the functional and basal layers, from 17 premenopausal (pre-MP) women, 19 post-menopausal (post-MP) women without hormonal treatment and 15 post-menopausal women on estrogen replacement therapy (post-MP+ E2), was collected through a prospective phase IV clinical trial over 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Post-menopausal women <65 years of age were treated with or without E2 for 6-8 weeks prior to tissue collection. Serum E2 levels were determined by estradiol immunoenzymatic assay. Endometrial tissue was obtained from women by biopsy (curettage) just prior to the hysterectomy. The effect of E2 on endometrial thickness and glandular and luminal epithelial height was determined using image analysis. Endometrial tissue was dissociated into single cell suspensions and MSC properties were examined in freshly isolated and short-term cultured, magnetic bead-purified W5C5(+) cells. MSC properties were assessed using clonogenicity, serial cloning, mesodermal differentiation in adipogenic, chondrogenic, osteogenic and myogenic induction culture media, and surface phenotype analysis by flow cytometry. Estrogen receptor α expression in W5C5(+) cells was examined using dual colour immunofluorescence. Vascularity was analysed using CD34 and alpha smooth muscle actin immunostaining and subsequent image analysis. MAIN RESULTS AND THE ROLE OF CHANCE A small population of stromal cells with MSC properties was purified with the W5C5 antibody from post-menopausal endometrium, whether atrophic from low circulating estrogen or regenerated from systemic estrogen treatment, similar to premenopausal endometrium. The MSC derived from post-menopausal endometrium treated with or without E2 fulfilled the minimum MSC criteria: clonogenicity, surface phenotype (CD29(+), CD44(+), CD73(+), CD105(+), CD140b(+), CD146(+)) and multipotency. The post-menopausal endometrial MSCs also showed comparable properties to premenopausal eMSC with respect to self-renewal in vitro and W5C5 expression. The W5C5(+) cells were located perivascularly as expected and did not express estrogen receptor α. LIMITATIONS, REASONS FOR CAUTION The properties of the MSC derived from post-menopausal endometrium were evaluated in vitro and their in vivo tissue reconstitution capacity has not been established as it has for premenopausal endometrial MSC. WIDER IMPLICATIONS OF THE FINDINGS The endometrium is an accessible source of MSC obtainable with minimum morbidity that could be used for future clinical applications as a cell-based therapy. This study shows that menopausal women can access their endometrial MSC by a simple biopsy for use in autologous therapies, particularly if their endometrium has been regenerated by short-term E2 treatment, provided they have an intact uterus and are not contraindicated for short-term E2 treatment. Endometrial MSC in post-menopausal women possess key MSC properties and are a promising source of MSC independent of a womans age. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Health and Medical Research Council (NHMRC) of Australia grant (1021126) (C.E.G., A.R.) and Senior Research Fellowship (1042298) (C.E.G.), Australian Gynaecological Endoscopic Society grant (A.R.) , Monash International Postgraduate Research Scholarship (DU), Australian Stem Cell Centre, South East Melbourne Alliance for Regenerative Therapies and Australian Stem Cell Centre top up scholarships (DU) and Victorian Governments Operational Infrastructure Support Program. Competing interests: AR receives Preceptorship fees from AMS, advisory board fees and sponsored study from Astellas, and conducts investigator led studies sponsored by AMS and Boston Scientific for other projects. TRIAL REGISTRATION NUMBER CTNRN12610000563066.

Isolation and characterisation of mesenchymal stem/stromal cells in the ovine endometrium.

Objective Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Materials and Methods Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. Results There was a small population CD271+ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271+CD49f- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271-CD49f- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271+ cells. Conclusion This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.

Management of the Placenta in Advanced Abdominal Pregnancies at an East African Tertiary Referral Center

OBJECTIVE To review the diagnosis and treatment of 9 advanced abdominal pregnancies in a low-resource setting of a developing country, focusing on the management of the placenta. METHODS Abdominal pregnancies occurring between 1999 and 2007 were identified from hospital records in Tanzania. All patients were followed up for a median of 6 months after surgery (range 5-9 months). RESULTS At the time of diagnosis, pregnancies were between 20 and 42 weeks of gestation (median 27 weeks). All 9 mothers survived the abdominal pregnancy, and 7 fetuses died before delivery. The placenta was left completely in situ in 5 of the nine cases. CONCLUSIONS Abdominal pregnancy is often detected rather late in low-resource settings compared with higher-resource settings. We suggest that in the described low-resource setting where red blood cell transfusions are not always readily available, the placenta may be left in situ after removal of the fetus.

Ten-Year Followup after Tension-Free Vaginal Tape-Obturator Procedure for Stress Urinary Incontinence

PURPOSE Suburethral tapes are a standard surgical treatment for stress urinary incontinence. The aim of the study was to evaluate subjective and objective cure rates 10 years after a tension-free vaginal tape-obturator procedure. MATERIALS AND METHODS All 124 patients who underwent the tension-free vaginal tape-obturator procedure at a total of 2 centers in 2004 and 2005 were invited for followup. Objective cure was defined as a negative cough stress test at 300 ml. Subjects completed KHQ (Kings Health Questionnaire), IOQ (Incontinence Outcome Questionnaire), FSFI (Female Sexual Function Index Questionnaire) and PGI-I (Patient Global Impression of Improvement). RESULTS Overall, 55 of 112 women (49%) who were alive were available for clinical examination and 71 (63%) completed the questionnaires. The objective cure rate in the 55 women examined clinically was 69%, 22% were not cured and 9% (5) had undergone reoperation for recurrent or persistent stress urinary incontinence. Treatment was counted as having failed in these 5 women for study purposes. Subjective cure was reported by 45 of 71 women (64%). Three patients (5%) had vaginal tape extrusion at the time of clinical examination. Extrusion in all of them was small and asymptomatic, and did not require treatment for a cumulative extrusion rate of 7%. Six women (9%) had undergone reoperation for tension-free vaginal tape-obturator associated complications and 18 (26%) experienced de novo overactive bladder. CONCLUSIONS Subjective and objective cure rates 10 years after the tension-free vaginal tape-obturator procedure were 69% and 64%, respectively. The vaginal extrusion rate in this study was slightly higher than in other series but major long-term complications appeared to be rare.