Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT Objective: To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey's HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

Tipos de partos y niveles de oxitocina, cortisol y hormonas tiroideas en plasma del cordón umbilical / Types of deliveries and levels of oxytocin, cortisol and thyroid hormones in umbilical cord plasma

Introducción: La placenta sintetiza y secreta varias hormonas que permiten la regulación del embarazo, el trabajo de parto y la adaptación metabólica materno-fetal. Su comportamiento asociado al tipo de parto puede dar información relevante sobre efectos epigenéticos. Objetivo: Describir el tipo de parto con los niveles de oxitocina, cortisol y hormonas tiroideas en plasma de cordón umbilical al nacer. Método: A 50 mujeres con embarazos principalmente normales se les cuantificaron los niveles neurohormonales en plasma de cordón umbilical, obtenido inmediatamente tras el periodo expulsivo. Los resultados se incorporaron a la base de datos clínicos de cada participante y se analizaron con Stata v.14.0. El protocolo fue aprobado por el comité de ética. Resultados: Hubo 33 partos vaginales (12 espontáneos, 13 acelerados y 8 inducidos) y 17 cesáreas (7 electivas y 10 de urgencia). Se observaron mayores niveles de cortisol en los partos vaginales acelerados; las cesáreas tuvieron menores niveles de cortisol y hormona estimulante de la tiroides. Las intervenciones clínicas, con altos o bajos niveles hormonales, están en directa relación con el tipo de parto. Conclusiones: El cortisol y la hormona estimulante de la tiroides medidos en plasma de cordón umbilical variaron según el tipo de parto. Esto es una primera cuantificación de hormonas en plasma de cordón umbilical y su posible regulación placentaria a propósito del tipo de parto.

Introduction: The placenta synthesizes and secretes several hormones allowing the regulation of pregnancy, labor and maternal-fetal metabolic adaptation. Their behavior associated with the type of delivery, may provide relevant information on epigenetic effects. Objective: To describe the type of delivery with the levels of oxytocin, cortisol and thyroid hormones in umbilical cord plasma at birth. Method: Neurohormonal levels from umbilical cord plasma obtained immediately post expulsion, were quantified in 50 women with mainly normal pregnancies. Results incorporated into the clinical database of each participant, statistically analyzed in Stata v.14.0. Protocol approved by ethics committee. Results: 33 were vaginal deliveries (12 spontaneous, 13 accelerated, 8 induced) and 17 cesarean sections (7 elective and 10 emergency). Higher cortisol levels were observed in accelerated vaginal deliveries, cesarean sections had lower cortisol and thyroid stimulating hormone levels. While clinical interventions, with high or low hormone levels, were related to the type of delivery. Conclusions: Cortisol and thyroid stimulating hormone measured in umbilical cord plasma varied according to the type of delivery. This is a first quantification of hormones in umbilical cord plasma and their possible placental regulation in relation to the type of delivery.

The haemodynamic effects of umbilical cord milking in term infants: a randomised controlled trial

INTRODUCTION@#Umbilical cord milking (UCM) is a method that allows for postnatal placental transfusion. The benefits of UCM have been demonstrated in some studies, but knowledge about its haemodynamic effects in term infants is limited. The aim of this study was to evaluate the haemodynamic effects of UCM in term infants.@*METHODS@#In this prospective, randomised controlled study, 149 healthy term infants with a birth week of ≥37 weeks were randomly assigned to either the UCM or immediate cord clamping (ICC) group. Blinded echocardiographic evaluations were performed in all the neonates in the first 2-6 h.@*RESULTS@#Superior vena cava (SVC) flow measurements were higher in the UCM group compared to the ICC group (132.47 ± 37.0 vs. 126.62 ± 34.3 mL/kg/min), but this difference was not statistically significant. Left atrial diameter (12.23 ± 1.99 vs. 11.43 ± 1.78 mm) and left atrium-to-aorta diastolic diameter ratio (1.62 ± 0.24 vs. 1.51 ± 0.22) were significantly higher in the UCM group. There were no significant differences in other echocardiographic parameters between the two groups.@*CONCLUSION@#We found no significant difference in the SVC flow measurements in term infants who underwent UCM versus those who underwent ICC. This lack of significant difference in SVC flow may be explained by the mature cerebral autoregulation mechanism in term neonates.

A prospective study on application of human umbilical cord mesenchymal stem cells combined with autologous Meek microskin transplantation in patients with extensive burns / 中华烧伤杂志

Objective: To investigate the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) combined with autologous Meek microskin transplantation on patients with extensive burns. Methods: The prospective self-controlled study was conducted. From May 2019 to June 2022, 16 patients with extensive burns admitted to the 990th Hospital of PLA Joint Logistics Support Force met the inclusion criteria, while 3 patients were excluded according to the exclusion criteria, and 13 patients were finally selected, including 10 males and 3 females, aged 24-61 (42±13) years. A total of 20 trial areas (40 wounds, with area of 10 cm×10 cm in each wound) were selected. Two adjacent wounds in each trial area were divided into hUCMSC+gel group applied with hyaluronic acid gel containing hUCMSCs and gel only group applied with hyaluronic acid gel only according to the random number table, with 20 wounds in each group. Afterwards the wounds in two groups were transplanted with autologous Meek microskin grafts with an extension ratio of 1∶6. In 2, 3, and 4 weeks post operation, the wound healing was observed, the wound healing rate was calculated, and the wound healing time was recorded. The specimen of wound secretion was collected for microorganism culture if there was purulent secretion on the wound post operation. In 3, 6, and 12 months post operation, the scar hyperplasia in wound was assessed using the Vancouver scar scale (VSS). In 3 months post operation, the wound tissue was collected for hematoxylin-eosin (HE) staining to observe the morphological changes and for immunohistochemical staining to observe the positive expressions of Ki67 and vimentin and to count the number of positive cells. Data were statistically analyzed with paired samples t test and Bonferronni correction. Results: In 2, 3, and 4 weeks post operation, the wound healing rates in hUCMSC+gel group were (80±11)%, (84±12)%, and (92±9)%, respectively, which were significantly higher than (67±18)%, (74±21)%, and (84±16)% in gel only group (with t values of 4.01, 3.52, and 3.66, respectively, P<0.05). The wound healing time in hUCMSC+gel group was (31±11) d, which was significantly shorter than (36±13) d in gel only group (t=-3.68, P<0.05). The microbiological culture of the postoperative wound secretion specimens from the adjacent wounds in 2 groups was identical, with negative results in 4 trial areas and positive results in 16 trial areas. In 3, 6, and 12 months post operation, the VSS scores of wounds in gel only group were 7.8±1.9, 6.7±2.1, and 5.4±1.6, which were significantly higher than 6.8±1.8, 5.6±1.6, and 4.0±1.4 in hUCMSC+gel group, respectively (with t values of -4.79, -4.37, and -5.47, respectively, P<0.05). In 3 months post operation, HE staining showed an increase in epidermal layer thickness and epidermal crest in wound in hUCMSC+gel group compared with those in gel only group, and immunohistochemical staining showed a significant increase in the number of Ki67 positive cells in wound in hUCMSC+gel group compared with those in gel only group (t=4.39, P<0.05), with no statistically significant difference in the number of vimentin positive cells in wound between the 2 groups (P>0.05). Conclusions: The application of hyaluronic acid gel containing hUCMSCs to the wound is simple to perform and is therefore a preferable route. Topical application of hUCMSCs can promote healing of the autologous Meek microskin grafted area in patients with extensive burns, shorten wound healing time, and alleviate scar hyperplasia. The above effects may be related to the increased epidermal thickness and epidermal crest, and active cell proliferation.

Effect of Hypoxia-Supported Umbilical Cord Mesenchymal Stem Cells on the Expansion of Cord Blood Mononuclear Cells in vitro / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of hypoxia-supported umbilical cord mesenchymal stem cell (UC-MSC) on the expansion of cord blood mononuclear cell （MNC） in vitro.@*METHODS@#The isolated cord blood mononuclear cells were inoculated on the preestablished umbilical cord mesenchymal stem cell layer and cultured under hypoxic conditions (3％ O2) and the experimental groups were normoxia (MNCs were cultured under normoxic conditions), hypoxia (MNCs were cultured under hypoxic conditions), UC-MSC (MNCs were cultured with UC-MSC under normoxic conditions), and UC-MSC+hypoxia (MNCs were cultured with UC-MSC under hypoxic conditions). To further investigate the combinational effect of 3 factors of SCF+FL+TPO (SFT) on expansion of cord blood MNCs in vitro in hypoxia-supported UC-MSC culture system, the experiments were further divided into group A (MNCs were cultured with UC-MSC and SFT under normoxic conditions), group B (MNCs were cultured with UC-MSC under hypoxic conditions), group C (MNCs were cultured with UC-MSC and SFT under hypoxic conditions). The number of nucleated cells (TNC), CD34+ cell, CFU and CD34+CXCR4+, CD34+CD49d+, CD34+CD62L+ cells of each groups were detected at 0, 7, 10 and 14 days, respectively.@*RESULTS@#Compared with group hypoxia and UC-MSC, group UC-MSC+hypoxia effectively promoted the expansion of TNC, CD34+ cell and CFU, and upregulated the expression level of adhesion molecule and CxCR4 of the cord blood CD34+ cell（P<0.05）. After culturing for 14 days, compared with group A and group B, group C effectively promoted the expansion of cord blood MNC at different time points（P<0.05）, and the effect of group A was better than that of group B at 7 and 10 days（P<0.05）.@*CONCLUSION@#Hypoxia-supported UC-MSC efficiently promoted the expansion and expression of adhesion molecule and CXCR4 of cord blood CD34+ cell, and the effect of expansion could be enhanced when SFT 3 factors were added.

Exosomes secreted from human umbilical cord mesenchymal stem cells promote pancreatic ductal adenocarcinoma growth by transferring miRNAs / 中华肿瘤杂志

Objective: To observe the effects of exosomes derived from human umbilical cord mesenchymal stem cells on the proliferation and invasion of pancreatic cancer cells, and to analyze the contents of exosomes and explore the mechanisms affecting pancreatic cancer cells. Methods: Exosomes extracted from human umbilical cord mesenchymal stem cells were added to pancreatic cancer cells BxPC3, Panc-1 and mouse models of pancreatic cancer, respectively. The proliferative activity and invasion abilities of BxPC3 and Panc-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The expressions of miRNAs in exosomes were detected by high-throughput sequencing. GO and KEGG were used to analyze the related functions and the main metabolic pathways of target genes with high expressions of miRNAs. Results: The results of CCK-8 cell proliferation assay showed that the absorbance of BxPC3 and Panc-1 cells in the hucMSCs-exo group was significantly higher than that in the control group [(4.68±0.09) vs. (3.68±0.01), P<0.05; (5.20±0.20) vs. (3.45±0.17), P<0.05]. Transwell test results showed that the number of invasion cells of BxPC3 and Panc-1 in hucMSCs-exo group was significantly higher than that in the control group (129.40±6.02) vs. (89.40±4.39), P<0.05; (134.40±7.02) vs. (97.00±6.08), P<0.05. In vivo experimental results showed that the tumor volume and weight in the exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) group were significantly greater than that in the control group [(884.57±59.70) mm(3) vs. (695.09±57.81) mm(3), P<0.05; (0.94±0.21) g vs. (0.60±0.13) g, P<0.05]. High-throughput sequencing results showed that miR-148a-3p, miR-100-5p, miR-143-3p, miR-21-5p and miR-92a-3p were highly expressed. GO and KEGG analysis showed that the target genes of these miRNAs were mainly involved in the regulation of glucosaldehylation, and the main metabolic pathways were ascorbic acid and aldehyde acid metabolism, which were closely related to the development of pancreatic cancer. Conclusion: Exosomes derived from human umbilical cord mesenchymal stem cells can promote the growth of pancreatic cancer cells and the mechanism is related to miRNAs that are highly expressed in exosomes.

Hernia Diafragmatica Izquierda. Reporte de un caso / Left Diaphragmatic Hernia: A case report

Se presenta un caso clínico de diagnóstico ecográfico de hernia diafragmática realizado a partir de signos de sospecha. La paciente se presenta a control prenatal cursando 18 semanas de gestación, donde se visualiza alteración de la posición de la silueta cardiaca y cambios en la ecogenicidad en el campo pulmonar izquierdo. Se evalúan los signos pronósticos de las hernias diafragmáticas como la presencia de estómago e hígado en tórax y se calcula el índice pulmóncabeza ( LHR). En el presente caso el LHR arrojo un valor de 2,3 %, lo cual indica un buen pronóstico ya que el LHR mayor a 1,4 % se asocia a 93 % de posibilidades de sobrevivir

A clinical case of ultrasound diagnosis of diaphragmatic hernia performed from signs of suspicion is presented. The patient is presented to prenatal control studying 18 weeks gestation, where alteration of the position of the cardiac silhouette and changes in ecogenicity in the left pulmonary field is displayed. The prognostic signs of diaphragmatic hernias such as the presence of stomach and thorax liver and the lung-headed index (LHR) are evaluated. In the present case, the LHR shows a value of 2.3 %, which indicates a good prognosis since the LHR greater than 1.4 % is associated with 93 % possibilities to survive

Hiperenrollamiento con torsión del cordón umbilical como causa de muerte fetal: reporte de caso / Hypercoiled umbilical cord torsion as a cause of fetal death: case report

Resumen La torsión del cordón umbilical como causa de muerte fetal es rara, con pocos casos reportados. No se conoce con claridad la causa y se presenta principalmente en el segundo trimestre de embarazo. Los factores de riesgo descritos son la longitud del cordón umbilical y el aumento del número de giros. Se reporta el caso de una paciente de 37 años, grávida 2, para 1 con embarazo de 23 semanas, con hallazgo ecográfico de muerte fetal. En el estudio de histopatología se evidenció el cordón umbilical con hiperenrollamiento y torsión a nivel de la unión feto-umbilical con oclusión de la luz de los vasos umbilicales como causa de muerte fetal. Se requiere la investigación de esta patología para determinar los factores de riesgo y el riesgo de recurrencia en futuros embarazos con el fin de establecer métodos de vigilancia fetal antenatal.

Abstract Torsion of the umbilical cord as a cause of fetal death is a rare occurrence, with few reported cases. The cause is not clearly known, and it transpires mainly in the second trimester of pregnancy; the risk factors described are the length of the umbilical cord with increased number of twists. The case of a 37-year-old woman is reported, gravida 2 para 1, 23 weeks pregnant with ultrasound diagnosis of fetal death. Histopathology revealed hypercoiled umbilical cord torsion at the point where the umbilical cord attaches to the fetus, with occlusion of the lumen of the umbilical vein, as a cause of fetal death. Further research of this pathology is required to determine the risk factors and risk of recurrence in future pregnancies that will allow the preparation of antenatal fetal surveillance methods.

Efficiency and safety of umbilical cord mesenchymal stromal/ stem cells in multiple sclerosis combined treatment / Eficacia y seguridad de las células madre / estromales mesenquimales del cordón umbilical en el tratamiento combinado de la esclerosis múltiple

SUMMARY: Multiple sclerosis is a demyelinating disease of the nervous system that affects young people of working age and quickly leads to disability. Treatment of this pathology with umbilical cord mesenchymal stem cells is promising, given their immunomodulatory and neurotrophic properties. The study involved 27 patients diagnosed with multiple sclerosis, 12 of whom underwent combined treatment (intravenous and intrathecal administration) of umbilical cord multipotent mesenchymal stromal/stem cells. The effectiveness of treatment was determined by the degree of neurological deficit and spasticity. Combined treatment with umbilical cord mesenchymal stem cells significantly improves the condition of patients with multiple sclerosis and promotes the regression of neurological deficits and spasticity. This treatment is safe, but for a deeper study, it is necessary to continue research in this area.

RESUMEN: La esclerosis múltiple es una enfermedad desmielinizante del sistema nervioso que afecta a los jóvenes en edad laboral y conduce rapidamente a la discapacidad. El tratamiento de esta patología con células madre mesenquimales de cordón umbilical es prometedor, debido a sus propiedades inmunomoduladoras y neurotróficas. En el estudio participaron 27 pacientes diagnosticados de esclerosis múltiple, 12 de los cuales fueron sometidos a un tratamiento combinado (administración intravenosa e intratecal) de células madre / estromales mesenquimales multipotentes del cordón umbilical. La efectividad del tratamiento estuvo determinada por el grado de déficit neurológico y espasticidad. El tratamiento combinado con células madre mesenquimales del cordón umbilical mejora significativamente la condición de los pacientes con esclerosis múltiple y promueve la regresión de los déficits neurológicos y la espasticidad. Este tratamiento es seguro, sin embargo, es necesario continuar investigando en esta area.

HucMSC-Ex alleviates inflammatory bowel disease via the lnc78583-mediated miR3202/HOXB13 pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

As a group of nonspecific inflammatory diseases affecting the intestine, inflammatory bowel disease (IBD) exhibits the characteristics of chronic recurring inflammation, and was proven to be increasing in incidence (Kaplan, 2015). IBD induced by genetic background, environmental changes, immune functions, microbial composition, and toxin exposures (Sasson et al., 2021) primarily includes ulcerative colitis (UC) and Crohn's disease (CD) with complicated clinical symptoms featured by abdominal pain, diarrhea, and even blood in stools (Fan et al., 2021; Huang et al., 2021). UC is mainly limited to the rectum and the colon, while CD usually impacts the terminal ileum and colon in a discontinuous manner (Ordás et al., 2012; Panés and Rimola, 2017). In recent years, many studies have suggested the lack of effective measures in the diagnosis and treatment of IBD, prompting an urgent need for new strategies to understand the mechanisms of and offer promising therapies for IBD.

Influence of umbilical cord milking versus delayed cord clamping on the early prognosis of preterm infants with a gestational age of <34 weeks: a Meta analysis / 中国当代儿科杂志

OBJECTIVES@#To study the influence of umbilical cord milking versus delayed cord clamping on the early prognosis of preterm infants with a gestational age of <34 weeks.@*METHODS@#PubMed, Web of Science, Embase, the Cochrane Library, CINAHL, China National Knowledge Infrastructure, Wanfang Data, Weipu Database, and SinoMed were searched for randomized controlled trials on umbilical cord milking versus delayed cord clamping in preterm infants with a gestational age of <34 weeks published up to November 2021. According to the inclusion and exclusion criteria, two researchers independently performed literature screening, quality evaluation, and data extraction. Review Manger 5.4 was used for Meta analysis.@*RESULTS@#A total of 11 articles were included in the analysis, with 1 621 preterm infants in total, among whom there were 809 infants in the umbilical cord milking group and 812 in the delayed cord clamping group. The Meta analysis showed that compared with delayed cord clamping, umbilical cord milking increased the mean blood pressure after birth (weighted mean difference=3.61, 95%CI: 0.73-6.50, P=0.01), but it also increased the incidence rate of severe intraventricular hemorrhage (RR=1.83, 95%CI: 1.08-3.09, P=0.02). There were no significant differences between the two groups in hemoglobin, hematocrit, blood transfusion rate, proportion of infants undergoing phototherapy, bilirubin peak, and incidence rates of complications such as periventricular leukomalacia and necrotizing enterocolitis (P>0.05).@*CONCLUSIONS@#Compared with delayed cord clamping, umbilical cord milking may increase the risk of severe intraventricular hemorrhage in preterm infants with a gestational age of <34 weeks; however, more high-quality large-sample randomized controlled trials are needed for further confirmation.

Human umbilical cord Wharton's jelly-derived mesenchymal stem cell transplantation could improve diabetic intracavernosal pressure / 亚洲男科学杂志(英文版)

Mesenchymal stem cells (MSCs) secrete various cytokines with angiogenic and neuroprotective effects. This study aimed to assess the effects of human umbilical cord Wharton's jelly-derived MSCs (hWJ-MSCs) on diabetes-related intracavernosal pressure (ICP) impairment in rats. hWJ-MSCs were isolated from human umbilical cord Wharton's jelly and transplanted into the corpus cavernosum of streptozotocin (STZ)-induced diabetic rats by unilateral injection. The erectile function was evaluated at 4 weeks, as well as the expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS), and insulin-like growth factor 1 (IGF1). STZ-induced diabetic rats showed impaired ICP, which was significantly improved by hWJ-MSC treatment. VEGF, eNOS, IGF1, and bFGF expression levels were higher in hWJ-MSC injection sites than those in control ones in STZ-induced diabetic rats. These results suggest that hWJ-MSC transplantation might improve diabetic erectile dysfunction through increased production of paracrine growth factors, highlighting a novel potential therapeutic option for erectile dysfunction.

Comparison of biological characteristics of placenta mesenchymal stem cells derived from fetus / 生物工程学报

Mesenchymal stem cells (MSCs) have broad application potentials in regenerative medicine and translational medicine. Obtaining large quantities of primary-cultured MSCs and select the most suitable cell origin for targeted diseases are critical to research. To select the most suitable seed cells of MSCs from different origins for clinical treatment and research, biological characteristics of MSCs from human umbilical cord and placenta were compared. These include cell morphology, surface marker expression, differentiation and karyotype. Transcriptome sequencing of four MSCs from fetus were performed and the results were analyzed from the perspective of proliferation and cytokine secretion. The results revealed that MSCs from umbilical cord (UC), amniotic membrane (AM), chorionic membrane (CM), chorionic villi (CV) and deciduae (DC) met the minimum standards of the International Society of Cell Therapy (ISCT) in 2006 and had the general characteristics of stem cells. Karyotype analysis showed that MSCs derived from UC, AM, CM and CV were all from fetus except that the DC-MSCs were from mother. Transcriptome sequencing analysis showed that hMSCs from umbilical cord and placenta had similar gene expression patterns, while different expression patterns were observed in specific genes involved in cell cycle, cell division, cell death, cell growth and development. These genes play important roles in transcriptional regulation, DNA repair, DNA replication and chromosome stability, which were momentous components of cellular or subcellular fraction movement, cell communication, cell tissue protrusions, cytokine secretion and hormone metabolism. Transcriptome sequencing analysis explained the differences in biological characteristics among MSCs from different sources, while verification experiments based on the transcriptome sequencing results showed that the proliferation and cytokine secretion capabilities of MSCs from different sources were significantly different. In all, UC-MSCs and CV-MSCs with stronger proliferation and higher levels of paracrine factors secretion may show their respective advantages in treating diseases.

Jujuboside A inhibits oxidative stress damage and enhances immunomodulatory capacity of human umbilical cord mesenchymal stem cells through up-regulating IDO expression / 中国天然药物

Impaired immunomodulatory capacity and oxidative stress are the key factors limiting the effectiveness of mesenchymal stem cell transplantation therapy. The present study was aimed to investigate the effects of jujuboside A (JuA) on the protective effect and immunomodulatory capacity of human umbilical cord mesenchymal stem cells (hUC-MSCs). Hydrogen peroxide was used to establish an oxidative damage model of hUC-MSCs, while PBMCs isolated from rats were used to evaluate the effect of JuA pre-treatment on the immunomodulatory capacity of hUC-MSCs. Furthermore, Hoechst 33258 staining, lactate dehydrogenase test, measurement of malondialdehyde, Western blot, high-performance liquid chromatography; and flow cytometry were performed. Our results indicated that JuA (25 μmol·L-1) promoted the proliferation of hUC-MSCs, but did not affect the differentiating capability of these cells. JuA pre-treatment inhibited apoptosis, prevented oxidative damage, and up-regulated the protein expression of nuclear factor-erythroid factor 2-related factor 2 and heme oxygenase 1 in hUC-MSCs in which oxidative stress was induced with H2O2. In addition, JuA pre-treatment enhanced the inhibitory effect of hUC-MSCs against abnormally activated PBMCs, which was related to stimulation of the expression and activity of indoleamine 2,3-dioxygenase. In conclusion, our results demonstrate that JuA pre-treatment can enhance the survival and immunomodulatory ability through pathways related to oxidative stress, providing a new option for the improvement of hUC-MSCs in the clinical setting.

Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

Factors associated with umbilical cord clamping in term newborns / Factores asociados al clampaje del cordón umbilical en recién nacidos a término / Fatores associados ao clampeamento do cordão umbilical em recém-nascidos a termo

ABSTRACT OBJECTIVE: To identify factors associated with umbilical cord clamping in term newborns and to compare the recording of clamping time in the medical record with what was observed. METHOD: Cross-sectional study, with 300 mothers-infants, in a university hospital. Clamping time and medical records were observed, and a structured questionnaire was applied to postpartum women for sociodemographic variables. Bivariate analysis, multivariate Poisson Regression model, and Kappa concordance test were performed. RESULTS: The percentage of late/optimal clamping observed was 53.7%. The associated factors were skin-to-skin contact in the delivery room (PR = 0.76; 0.61-0.95; p = 0.014), position of the newborn below the vaginal canal (PR = 2.6; CI95%: 1.66-4.07; p < 0.001), position of the newborn at the vaginal level (PR = 2.03; CI95%: 1.5-2.75; p < 0.001), and need for newborn resuscitation in the delivery room (PR = 1.42; CI95%; 1.16-1.73; p = 0.001). Kappa concordance level of the professionals, records compared to the observation was: nurse 0.47, obstetrician 0.59, and pediatrician 0.86. CONCLUSION: the identification of associated factors and the comparison between recording and observing the clamping time can help in the planning and implementation of improvements for adherence to good practices at birth.

RESUMEN Objetivo: Identificar factores asociados al clampaje del cordón umbilical en neonatos a término y comparar el registro del tiempo de clampaje en el prontuario con el observado. Método: Estudio transversal, con 300 madres-hijos, en hospital universitario. Se observó tiempo de clampaje y registros en prontuario, siendo aplicado cuestionario estructurado a las puérperas para variables sociodemográficas. Se utilizó análisis bivariado, modelo de Regresión de Poisson multivariado e índice de concordancia Kappa. Resultados: el porcentaje de clampaje tardío/oportuno observado fue 53,7%. Los factores asociados fueron contacto piel con piel en sala de partos (RP = 0,76; 0,61-0,95; p = 0,014), posición del recién nascido abajo del canal vaginal (RP = 2,6; IC95%: 1,66-4,07; p < 0,001), posición del recién nacido en el nivel vaginal (RP = 2,03; IC95%: 1,5-2,75; p < 0,001) y necesidad de reanimación del recién nacido en sala de partos (RP = 1,42; IC95%; 1,16-1,73; p = 0,001). El nivel de concordancia Kappa del registro de los profesionales comparado a la observación fue: enfermera 0,47, obstetra 0,59 y pediatra 0,86. Conclusión: la identificación de los factores asociados y la comparación entre registro y observación del tiempo de clampaje puede auxiliar en la planificación e implementación de mejorías para la adhesión a las buenas prácticas de nacimiento.

RESUMO Objetivo: identificar fatores associados ao clampeamento do cordão umbilical em neonatos a termo e comparar o registro do tempo de clampeamento no prontuário com o observado. Método: estudo transversal, com 300 mães-bebês, em hospital universitário. Observaram-se tempo de clampeamento e registros em prontuário, sendo aplicado questionário estruturado às puérperas para variáveis sociodemográficas. Procedeu-se análise bivariada, modelo de Regressão de Poisson multivariado e teste de concordância Kappa. Resultados: o percentual de clampeamento tardio/oportuno observado foi 53,7%. Os fatores associados foram contato pele-a-pele em sala de parto (RP = 0,76; 0,61-0,95; p = 0,014), posição do recém-nascido abaixo do canal vaginal (RP = 2,6; IC95%: 1,66-4,07; p < 0,001), posição do recém-nascido no nível vaginal (RP = 2,03; IC95%: 1,5-2,75; p < 0,001) e necessidade de reanimação do recém-nascido em sala de parto (RP = 1,42; IC95%; 1,16-1,73; p = 0,001). O nível de concordância Kappa do registro dos profissionais comparado à observação foi: enfermeira 0,47, obstetra 0,59 e pediatra 0,86. Conclusão: a identificação dos fatores associados e a comparação entre registro e observação do tempo de clampeamento podem auxiliar no planejamento e implementação de melhorias para adesão às boas práticas no nascimento.

Recovery of motor function in rats with complete spinal cord injury following implantation of collagen/silk fibroin scaffold combined with human umbilical cord-mesenchymal stem cells

SUMMARY OBJECTIVE: This study aimed to assess the effect of the collagen/silk fibroin scaffolds seeded with human umbilical cord-mesenchymal stem cells on functional recovery after acute complete spinal cord injury. METHODS: The fibroin and collagen were mixed (mass ratio, 3:7), and the composite scaffolds were produced. Forty rats were randomly divided into the Sham group (without spinal cord injury), spinal cord injury group (spinal cord transection without any implantation), collagen/silk fibroin scaffolds group (spinal cord transection with implantation of the collagen/silk fibroin scaffolds), and collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (spinal cord transection with the implantation of the collagen/silk fibroin scaffolds co-cultured with human umbilical cord-mesenchymal stem cells). Motor evoked potential, Basso-Beattie-Bresnahan scale, modified Bielschowsky's silver staining, and immunofluorescence staining were performed. RESULTS: The BBB scores in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group were significantly higher than those in the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). The amplitude and latency were markedly improved in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). Meanwhile, compared to the spinal cord injury and collagen/silk fibroin scaffolds groups, more neurofilament positive nerve fiber ensheathed by myelin basic protein positive structure at the injury site were observed in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (p<0.01, p<0.05). The results of Bielschowsky's silver staining indicated more nerve fibers was observed at the lesion site in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.01, p< 0.05). CONCLUSION: The results demonstrated that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen/silk fibroin scaffolds could promote nerve regeneration, and recovery of neurological function after acute spinal cord injury.

Ligadura del cordón umbilical en recién nacidos prematuros / Umbilical cord clamping in preterm infants

La prematiridad fue aumentando la supervivencia desde hace varios años, y eso produce, sobre todo, una preocupación en los prematuros nacidos antes de las 28 semanas de gestación. El tiempo del clampeo del cordón umbilical puede generar diversos trastornos, principalmente, cuando se realiza temprano (10-15 segundos). Ya desde hace 20 años, a través de varias investigaciones, se pudieron demostrar los notorios beneficios del clampeo demorado del cordón (de 2 a 3 minutos). Esta práctica fue instalada en la asistencia obstétrica y neonatal por las recomendaciones de sociedades científicas y de las revisiones sistemáticas, que señalaron las sólidas evidencias que apoyaban esta conducta para prematuros. En esta revisión, se describen los artículos más relevantes en los últimos años, que sustentan notoriamente la aplicación del clampeo demorado del cordón versus el clampeo temprano. Asimismo, esta práctica genera una disminución de los trastornos graves en prematuros.

For several years now, the survival of preterm infants has been increasing, which has shifted our concern to preterm infants born before 28 weeks of gestation in particular. The timing of umbilical cord clamping may lead to several disorders, especially when done early (10-15 seconds). In the last two decades, several investigations have shown the considerable benefits of delayed cord clamping (2-3 minutes). Delayed cord clamping has been practiced in obstetrics and neonatal care based on the recommendations made by scientific societies and in systematic reviews, which have provided solid evidence to support this practice in preterm infants. This review describes the most relevant articles from the last years, which strongly support the use of delayed cord clamping versus early cord clamping. In addition, this practice reduces the rate of severe disorders in preterm infants.

Diagnóstico posnatal de hematoma de cordón umbilical: reporte de un caso / Postnatal diagnosis of umbilical cord hematoma: case report

ANTECEDENTES: El hematoma espontáneo del cordón umbilical es una complicación rara del embarazo que representa una causa grave de morbilidad y mortalidad fetal. Se han descrito numerosos factores de riesgo, pero aún se desconoce su etiología exacta. CASO CLÍNICO: Mujer de 28 años, en su octava gestación, que consultó en urgencias por disminución de los movimientos fetales de varias horas de evolución tras una caída accidental traumática. La monitorización fetal mostró un patrón cardiotocográfico no tranquilizador, con disminución de la variabilidad y sin aceleraciones. Se realizó una cesárea urgente, sin complicaciones, con el nacimiento de un varón vivo de 4560 g, con Apgar 8/10/10 y pH de sangre de cordón umbilical 7.08, que precisó ingreso en la unidad de neonatología por hallazgo de un soplo cardíaco. Tras el alumbramiento se objetivó una gran colección hemática en el cordón umbilical. MÉTODO: Se aplicó una estrategia de búsqueda sistemática en Medline, PubMed y Cochrane de todos los artículos en inglés y español que tuvieran como palabras clave "Umbilical", "Cordón" y "Hematoma". RESULTADOS: Se encontraron 13 publicaciones de 15 casos de hematoma del cordón umbilical en los años 2008-2020. Se realizó una revisión sistemática de los informes de casos descritos en los últimos 12 años en la literatura para evaluar la epidemiología, los factores predisponentes, los resultados potenciales, el diagnóstico prenatal y el manejo clínico. CONCLUSIONES: Debido a la rareza de esta afección, se deben informar todos los casos nuevos de hematoma del cordón umbilical para mejorar el conocimiento de los factores predisponentes, el diagnóstico prenatal y el manejo clínico.

BACKGROUND: Spontaneous umbilical cord hematoma is a rare complication of pregnancy which represents a serious cause of fetal morbidity and mortality. There are many risk factors, but the exact etiology is still unknown. CASE REPORT: 28-year-old woman, eighth gestation, who consulted due to decreased fetal movements of several hours of evolution after traumatic accidental fall. Fetal monitoring showed a non-reassuring cardiotocographic pattern, with decreased fetal variability, without accelerations. An emergency cesarean section was performed without complications, with the birth of a living male weighing 4560 grams, with an 8/10/10 Apgar test, and an arterial pH of the umbilical cord 7.08, which required admission to the neonatology unit. After delivery, a large blood collection was observed in the umbilical cord. METHOD: A systematic search strategy was applied to several electronic bibliographic databases: Medline, PubMed and Cochrane. Key words used were "Umbilical", "Cord", "Hematoma". RESULTS: 13 publications of 15 cases of umbilical cord hematoma were reported in the years 2008-2020. A systematic review of the reports of cases, described in the last twelve years in the literature was carried out to evaluate the epidemiology, predisposing factors, potential results, prenatal diagnosis and clinical management of this phenomenon. CONCLUSIONS: Due to the rarity of this condition, every new case of umbilical cord hematoma should be reported in order to improve the knowledge of predisposing factors, prenatal diagnosis, and clinical management.