Cell lines and their passages used in this study.

*<p>The passages, indicating for how long hPSCs were cultured in SNL and Matrigel conditions, are given in parenthesis.</p><p>TNE  =  Total number of experiments.</p><p>TNW  =  Total number of wells from which the beating areas were counted.</p

Culture Conditions Affect Cardiac Differentiation Potential of Human Pluripotent Stem Cells

<div><p>Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are capable of differentiating into any cell type in the human body and thus can be used in studies of early human development, as cell models for different diseases and eventually also in regenerative medicine applications. Since the first derivation of hESCs in 1998, a variety of culture conditions have been described for the undifferentiated growth of hPSCs. In this study, we cultured both hESCs and hiPSCs in three different culture conditions: on mouse embryonic fibroblast (MEF) and SNL feeder cell layers together with conventional stem cell culture medium containing knockout serum replacement and basic fibroblast growth factor (bFGF), as well as on a Matrigel matrix in mTeSR1 medium. hPSC lines were subjected to cardiac differentiation in mouse visceral endodermal-like (END-2) co-cultures and the cardiac differentiation efficiency was determined by counting both the beating areas and Troponin T positive cells, as well as studying the expression of <em>OCT-3/4</em>, mesodermal <em>Brachyury T</em> and <em>NKX2.5</em> and endodermal <em>SOX-17</em> at various time points during END-2 differentiation by q-RT-PCR analysis. The most efficient cardiac differentiation was observed with hPSCs cultured on MEF or SNL feeder cell layers in stem cell culture medium and the least efficient cardiac differentiation was observed on a Matrigel matrix in mTeSR1 medium. Further, hPSCs cultured on a Matrigel matrix in mTeSR1 medium were found to be more committed to neural lineage than hPSCs cultured on MEF or SNL feeder cell layers. In conclusion, culture conditions have a major impact on the propensity of the hPSCs to differentiate into a cardiac lineage.</p> </div

The expression of neural markers was highest in hPSCs originating from Matrigel.

<p>A) H7 cell line was hard to adapt on Matrigel combined with mTeSR1 medium. At first, the neural differentiation was observed primarily along the edges of the colonies on Matrigel in mTeSR1 medium. Finally, uneven neural rosette-like structures were formed in the colonies and the cell line was lost. B) Uneven neural rosette-like structures found in colonies of H7 cell line cultured on Matrigel in mTeSR1 medium stained with MAP-2. C) MAP-2 expressing neural-like cells and structures were found on Matrigel in mTeSR1 medium in all hPSC lines. Representative image of UTA.04602.WT cell line. D) MAP-2 expressing neural structures appeared also in END-2 co-cultures when hPSCs originated from Matrigel and mTeSR1 cultures with all hPSC lines. Representative image of H7 cell line. Scale bars, 200 μm. E) The highest amount of MAP-2 positive cells were found in Matrigel cultures. Scale bars, 200 μm. The expression of <i>PAX-6 (F), Musashi (G)</i> and <i>Neurofilament (NF-68)</i> (H) in END-2 co-cultures was significantly higher in cells originating from Matrigel and mTeSR1 medium than from MEF or SNL feeder cell layers almost in all time points. The data is collected from two individual differentiation experiments of H7, UTA.00112.hFF and UTA.04602.WT hPSC lines (n = 6 in all three conditions). Error bars show the standard error of the mean (SEM). ** p<0.01, * p<0.05.</p

hPSCs were cultured with three different culture methods.

<p>A) Schematic presentation of cardiac differentiation in END-2 co-culture and the experimental design. hPSC  =  human pluripotent stem cell, END-2 =  mouse visceral endodermal-like cells. Scale bars, 200 μm. B) All five hPSC lines cultured on MEF and SNL feeder cell layers in conventional stem cell culture medium and on Matrigel in mTeSR1 medium at least for 14 passages formed undifferentiated colonies, which expressed pluripotency markers Nanog, OCT-3/4 and SSEA-4. Representative images of UTA.00112.hFF (phase contrast microscope images) and H7 (immunofluorescence images) cell lines are presented. Scale bars, 200 μm. C) H7, UTA.00106.hFF and UTA.00525.LQT2 cell lines cultured in all three culture conditions (in figure: first band MEF, second band SNL, third band Matrigel) formed embryoid bodies (EBs) expressing markers from all germ layers: ectoderm (<i>PAX-6</i> and <i>SOX-1</i>), endoderm (<i>AFP</i> and <i>SOX-17</i>) and mesoderm (<i>α-cardiac actin</i> and <i>KDR</i>).</p

The amount of TRA-1-81 and PSA-NCAM positive cells in hPSC cultures.

<p>A) The amount of TRA-1-81 positive cells was higher and the expression of PSA-NCAM positive cells was lower in MEF feeder cultures than in SNL or Matrigel cultures. Columns show the average of TRA-1-81 and PSA-NCAM positive cells of all hPSC lines cultured in three different conditions (MEF n = 16, SNL n = 20 and Matrigel n = 15). Error bars show the standard error of the mean (SEM). B) The amount of TRA-1-81 and PSA-NCAM cells for each hPSC lines cultured in all three conditions. C) Examples of TRA-1-81 and PSA-NCAM expressions in H7 cell line in all three conditions. Dot plots show the determination of hPSC population and histograms show the percentage of TRA-1-81 and PSA-NCAM positive cells. Unstained cells were used for background determination (white). The highest amount of PSA-NCAM positive cells in immunocytochemical stainings were found on Matrigel. PSA-NCAM positive cells were not detected on MEF feeder cell cultures. Scale bars, 200 μm.</p

Overview of published population-based studies assessing the prevalence of overactive bladder (OAB^*) among both sexes (MEDLINE and PubMed search to December 2006) with present study (<i>in chronological order</i>)

*<p>OAB, overactive bladder; UTI, urinary tract infection</p>†<p>In the European study, in five out of six countries, telephone interview was used (excluding Spain where direct interviews were conducted due to lower proportion of households having telephone)</p>‡<p>Out of 11,740 participants (of 17,231 households contacted), 5,539 were considered ineligible. To calculate response rate, the number of respondents was divided by eligible participants (<i>the former response rate</i>). If same proportion of non-participants, as there were ineligible among participants (47%), were also considered ineligible, response rate was greater (<i>the latter response rate</i>).</p>§<p>Study sample was close to representative of the general population regarding age, and/or age-standardization was used.</p

Figure 2

<p>The prevalence of overactive bladder in Finland, 2003–2004. The blue bars indicate men with overactive bladder and the red bars women with overactive bladder. Age-standardization was performed using the general population <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000195#pone.0000195-The1" target="_blank">[21]</a>.</p

Exclusions of the study population of overactive bladder analysis: number of excluded subjects among 1725 men and 2002 women in Finland, 2003–2004

*<p>Acute (in past 2 weeks) or chronic urinary tract infection.</p>†<p>Excluding renal cancer.</p>‡<p>Due to e.g. painful bladder syndrome or radiation.</p>§<p>Puerperium defined as 6 weeks after childbirth.</p

Figure 3

<p>Age-standardized prevalence of overactive bladder symptoms among Finnish people aged 18–79 years, 2003–2004. The red circle represents subjects with overactive bladder without urgency incontinence excluding the area of the red oval representing subjects with overactive bladder with urgency incontinence. The blue circle represents subjects with urinary frequency (defined as more than eight voids per day) and the green circle nocturia (defined as more than one void per night). Age-standardization was performed using the general population <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000195#pone.0000195-The1" target="_blank">[21]</a>.</p

Table_1_Association of concomitant autoimmunity with the disease features and long-term treatment and health outcomes in Celiac disease.DOCX

BackgroundCeliac disease (CeD) is often accompanied by other autoimmune diseases (AID). However, the association of co-existing autoimmunity with the presentation and treatment success in CeD is unclear. We investigated these issues with a large and well-defined cohort of Finnish patients.MethodsAdult CeD patients (n = 806) were collected from multiple heath care sites via nationwide recruitment. They were interviewed, underwent measurement of CeD autoantibodies, and filled out questionnaires to ascertain quality of life (PGWB) and gastrointestinal symptoms (GSRS) after a median of 9.7 years on a gluten-free diet. Data were supplemented retrospectively from patient records. The results were compared between CeD patients with and without a coexisting AID.ResultsAltogether 185 patients had CeD+AID and 621 had CeD only. At CeD diagnosis, patients with CeD+AID were older (median 42 vs. 36 years, p = 0.010) and had more joint symptoms (9.1 vs. 4.2%, p = 0.011), whereas the groups were comparable in sex, family history of CeD, other presenting symptoms, proportion of screen-detected subjects, and severity of duodenal lesion. During follow-up on gluten-free diet, CeD+AID patients experienced poorer general health (median score 12 vs. 14, p ConclusionsCo-existing AID was not significantly associated with the baseline features or with most long-term outcomes in CeD. However, the increased prevalence of gastrointestinal symptoms and reduced poorer self-perceived health during treatment indicates these patients' need for special support.</p