Term Umbilical Cord Research Articles

Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant

BackgroundBleomycin (BLM)-induced lung injury is characterized by mixed histopathologic changes with inflammation and fibrosis, such as observed in human patients with bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Although no curative therapies for these lung diseases exist, stem cell therapy has emerged as a potential therapeutic option. Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent- and macrophage-like stem cells distributed in various adult and fetal tissues as stage-specific embryonic antigen-3-positive cells. They selectively home to damaged tissue by sensing sphingosine-1-phosphate and replace the damaged/apoptotic cells by in vivo differentiation. Clinical trials for some human diseases suggest the safety and therapeutic efficacy of intravenously injected human leukocyte antigen-mismatched allogenic Muse cells from adult bone marrow (BM) without immunosuppressant. Here, we evaluated the therapeutic effects of human Muse cells from preterm and term umbilical cord (UC), and adult BM in a rat BLM-induced lung injury model.MethodsRats were endotracheally administered BLM to induce lung injury on day 0. On day 3, human preterm UC-Muse, term UC-Muse, or adult BM-Muse cells were administered intravenously without immunosuppressants, and rats were subjected to histopathologic analysis on day 21. Body weight, serum surfactant protein D (SP-D) levels, and oxygen saturation (SpO2) were monitored. Histopathologic lung injury scoring by the Ashcroft and modified American Thoracic Society document scales, quantitative characterization of engrafted Muse cells, RNA sequencing analysis, and in vitro migration assay of infused Muse cells were performed.ResultsRats administered preterm- and term-UC-Muse cells exhibited a significantly better recovery based on weight loss, serum SP-D levels, SpO2, and histopathologic lung injury scores, and a significantly higher rate of both Muse cell homing to the lung and alveolar marker expression (podoplanin and prosurfactant protein-C) than rats administered BM-Muse cells. Rats receiving preterm-UC-Muse cells showed statistically superior results to those receiving term-UC-Muse cells in many of the measures. These findings are thought to be due to higher expression of genes related to cell migration, lung differentiation, and cell adhesion.ConclusionPreterm UC-Muse cells deliver more efficient therapeutic effects than term UC- and BM-Muse cells for treating BLM-induced lung injury in a rat model.

Characterization of Angiogenic, Matrix Remodeling, and Antimicrobial Factors in Preterm and Full-Term Human Umbilical Cords.

Little is known about morphogenetic changes in the umbilical cord during the maturation process. Extracellular matrix remodeling, angiogenesis, progenitor activity, and immunomodulation are represented by specific markers; therefore, the aim of this study was to determine the expression of matrix metalloproteinase-2 (MMP2), tissue inhibitor of metalloproteinases-2 (TIMP2), CD34, vascular endothelial growth factor (VEGF), and human β-defensin 2 (HBD2) in preterm and full-term human umbilical cord tissue. Samples of umbilical cord tissue were obtained from 17 patients and divided into two groups: very preterm and moderate preterm birth umbilical cords; late preterm birth and full-term birth umbilical cords. Routine histology examination was conducted. Marker-positive cells were detected using the immunohistochemistry method. The number of positive structures was counted semi-quantitatively using microscopy. Statistical analysis was carried out using the SPSS Statistics 29 program. Extraembryonic mesenchyme cells are the most active cell producers, expressing MMP2, TIMP2, VEGF, and HBD2 at notable levels in preterm and full-term umbilical cord tissue. Statistically significant differences in the expression of CD34, MMP2, and TIMP2 between the two patient groups were found. The expression of VEGF was similar in both patient groups, with the highest number of VEGF-positive cells seen in the extraembryonic mesenchyme. The expression of HBD2 was the highest in the extraembryonic mesenchyme and the amniotic epithelium, where mostly moderate numbers of HBD2-positive cells were detected. Extracellular matrix remodeling in preterm and term umbilical cords is strongly regulated, and tissue factors MMP2 and TIMP2 take part in this process. The expression of VEGF is not affected by the umbilical cord's age; however, individual patient factors can affect the production of VEGF. Numerous CD34-positive cells in the endothelium of the umbilical arteries suggest a significant role of progenitor cells in very preterm and moderate preterm birth umbilical cords. Antimicrobial activity provided by HBD2 is essential and constant in preterm and full-term umbilical cords.

Long Non-Coding RNA H19 Leads to Upregulation of γ-Globin Gene Expression during Erythroid Differentiation

Long noncoding RNAs (lncRNAs) are important because they are involved in a variety of life activities and have many downstream targets. Moreover, there is also increasing evidence that some lncRNAs play important roles in the expression and regulation of γ-globin genes. In our previous study, we analyzed genetic material from nucleated red blood cells (NRBCs) extracted from premature and full-term umbilical cord blood samples. Through RNA sequencing (RNA-Seq) analysis, lncRNA H19 emerged as a differentially expressed transcript between the two blood types. While this discovery provided insight into H19, previous studies had not investigated its effect on the γ-globin gene. Therefore, the focus of our study was to explore the impact of H19 on the γ-globin gene. In this study, we discovered that overexpressing H19 led to a decrease in HBG mRNA levels during erythroid differentiation in K562 cells. Conversely, in CD34+ hematopoietic stem cells and human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, HBG expression increased. Additionally, we observed that H19 was primarily located in the nucleus of K562 cells, while in HUDEP-2 cells, H19 was present predominantly in the cytoplasm. These findings suggest a significant upregulation of HBG due to H19 overexpression. Notably, cytoplasmic localization in HUDEP-2 cells hints at its potential role as a competing endogenous RNA (ceRNA), regulating γ-globin expression by targeting microRNA/mRNA interactions.

Banked term umbilical cord blood to meet the packed red blood cell transfusion needs of extremely-low-gestational-age neonates: a feasibility analysis.

To assess the feasibility of drawing, processing, safety-testing, and banking term umbilical cord blood to meet the packed red blood cell transfusion (RBC Tx) needs of extremely-low-gestational-age neonates (ELGANs). (1) Retrospectively analyze all ELGANs RBC Tx over the past three years, (2) Estimate local cord blood availability, (3) Assess interest in this project, and implementation barriers, through stakeholder surveys. In three years we cared for 266 ELGANs; 165 (62%) received ≥1 RBC Tx. Annual RBC Tx averaged 197 (95% CI, 152-243). If 10% of our 10,353 annual term births had cord blood drawn and processed, and half of those tested were acceptable for Tx, collections would exceed the 95th % upper estimate for need by >four-fold. Interest exceeded 97%. Identified barriers included FDA approval, training to collect cord blood, and cost. RBC Tx needs of ELGANS could be met by local cord blood collection.

Neonatal immune cells have heightened responses following in-utero exposure to chorioamnionitis or COVID-19.

Chorioamnionitis alters neonatal immune responses. Gestational COVID-19 infection is associated with adverse pregnancy outcomes, but its impact on neonatal immunity is unclear. We hypothesized that gestational COVID-19 exposure would result in exaggerated neonatal immune responses, similar to chorioamnionitis-exposed neonates. Term umbilical cord blood mononuclear cells (CBMCs) were isolated from neonates exposed to chorioamnionitis, gestational COVID-19 or unexposed controls. CBMCs were cultured and stimulated with heat-killed Escherichia coli, Streptococcus agalactiae or Staphylococcus epidermidis. A multiplexed protein assay was used to measure cytokine levels in cell culture supernatants and flow cytometry was used to evaluate cellular-level cytokine expression. Both chorioamnionitis-exposed and COVID-19 exposed CBMCs demonstrated upregulation of IL-1β and IL-6 compared to unexposed CBMCs, while only COVID-19 exposure resulted in IL-8 upregulation. There were no differences between chorioamnionitis-exposed and COVID-19 exposed CBMCs when these groups were directly compared. Flow cytometry demonstrated immune cell subset specific differences in cytokine expression between the exposure groups. The fetal/neonatal response to maternal inflammation differed based on immune cell subset and etiology of inflammation, but the global neonatal cytokine responses were similar between exposure groups. This suggests that targeting perinatal inflammation rather than the specific etiology may be a possible therapeutic approach. Neonatal immune cells have similar pathogen-associated global cytokine responses, but different cell-level immune responses, following in-utero exposure to chorioamnionitis or COVID-19. This is the first study to directly compare immune responses between neonates exposed to chorioamnionitis and COVID-19. This suggests that the fetal/neonatal cellular response to perinatal inflammation differs based on the etiology and severity of maternal inflammation, but still results in a similar overall inflammatory profile regardless of the cause of perinatal inflammation.

Peptidome analysis of umbilical cord mesenchymal stem cell (hUC-MSC) conditioned medium from preterm and term infants

BackgroundThe therapeutic role of mesenchymal stem cells (MSCs) has been widely confirmed in several animal models of premature infant diseases. Micromolecule peptides have shown promise for the treatment of premature infant diseases. However, the potential role of peptides secreted from MSCs has not been studied. The purpose of this study is to help to broaden the knowledge of the hUC-MSC secretome at the peptide level through peptidomic profile analysis.MethodsWe used tandem mass tag (TMT) labeling technology followed by tandem mass spectrometry to compare the peptidomic profile of preterm and term umbilical cord MSC (hUC-MSC) conditioned medium (CM). Gene Ontology (GO) enrichment analysis and ingenuity pathway analysis (IPA) were conducted to explore the differentially expressed peptides by predicting the functions of their precursor proteins. To evaluate the effect of candidate peptides on human lung epithelial cells stimulated by hydrogen peroxide (H2O2), quantitative real-time PCR (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were, respectively, adopted to detect inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression levels at the mRNA and protein levels.ResultsA total of 131 peptides derived from 106 precursor proteins were differentially expressed in the preterm hUC-MSC CM compared with the term group, comprising 37 upregulated peptides and 94 downregulated peptides. Bioinformatics analysis showed that these differentially expressed peptides may be associated with developmental disorders, inflammatory response, and organismal injury. We also found that peptides 7118TGAKIKLVGT7127 derived from MUC19 and 508AAAAGPANVH517 derived from SIX5 reduced the expression levels of TNF-α, IL-1β, and IL-6 in H2O2-treated human lung epithelial cells.ConclusionsIn summary, this study provides further secretomics information on hUC-MSCs and provides a series of peptides that might have antiinflammatory effects on pulmonary epithelial cells and contribute to the prevention and treatment of respiratory diseases in premature infants.

Oxygen Disrupts Human Fetal Lung Mesenchymal Cells. Implications for Bronchopulmonary Dysplasia.

Exogenous mesenchymal stromal cells (MSCs) ameliorate experimental bronchopulmonary dysplasia. Moreover, data from term-born animal models and human tracheal aspirate-derived cells suggest altered mesenchymal signaling in the pathophysiology of neonatal lung disease. We hypothesized that hyperoxia, a factor contributing to the development of bronchopulmonary dysplasia, perturbs human lung-resident MSC function. Mesenchymal cells were isolated from human fetal lung tissue (16-18 wk of gestation), characterized and cultured in conditions resembling either intrauterine (5% O2) or extrauterine (21% and 60% O2) atmospheres. Secretome data were compared with MSCs obtained from term umbilical cord tissues. The human fetal lung mesenchyme almost exclusively contains CD146pos. MSCs expressing SOX-2 and OCT-4, which secrete elastin, fibroblast growth factors 7 and 10, vascular endothelial growth factor, angiogenin, and other lung cell-protecting/-maturing proteins. Exposure to extrauterine atmospheres in vitro leads to excessive proliferation, reduced colony-forming ability, alterations in the cell's surface marker profile, decreased elastin deposition, and impaired secretion of factors important for lung growth. Conversely, umbilical cord-derived MSCs abundantly secreted factors that impaired lung MSCs are unable to produce. Oxygen-impaired human fetal lung MSC function may contribute to disrupted repair capacity and arrested lung growth. Exogenous MSCs may act by triggering the signaling pathways lost by impaired endogenous lung mesenchymal cells.

A pilot study: calciprotein particle levels in term umbilical cord blood at delivery

A pilot study: calciprotein particle levels in term umbilical cord blood at delivery

Stem cells derived from human first-trimester umbilical cord have the potential to differentiate into oocyte-like cells in vitro

Compared to stem cells derived from human term umbilical cord, stem cells derived from human first-trimester umbilical cord (hFTUC) exhibit a significantly greater proliferative potential, and more efficiency in terms of their in vitro differentiation. In the present study, we investigated whether hFTUC-derived stem cells are able to differentiate into germ cells. The hFTUC-derived stem cells were first isolated, expanded and then cultured in differentiation medium containing human follicular fluid, follicle-stimulating hormone (FSH)/luteinizing hormone (LH) and estradiol for 24 days. During the period of induction, a subpopulation of the cultured cells appeared that had a morphological resemblance to primordial germ cells (PGCs) and cumulus-oocyte complex (COC)-like cells, and oocyte-like cells (OLCs). The PGC-like cells expressed specific markers indicative of germ cell formation such as octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen 1 (SSEA1), B lymphocyte-induced maturation protein-1 (BLIMP1), PR domain containing 14 (PRDM14), transcription factor AP-2 gamma (TFAP2C), VASA, STELLA, deleted in azoospermia-like (DAZL) and interferon-induced transmembrane protein 3 (IFITM3). The OLCs, which contained a single germinal vesicle, expressed oocyte-specific markers, such as synaptonemal complex protein 3 (SCP3), growth/differentiation factor-9 (GDF9), GDF9B and zona pellucida (ZP)1, ZP2 and ZP3. The COC-like cells secreted estradiol, vascular endothelial growth factor and leukemia inhibitory factor. Thus, our findings suggest that hFTUC-derived stem cells have an intrinsic ability to differentiate into OLCs, which may provide an in vitro model for the identification of factors involved in germ cell formation and differentiation.

Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure

Background aimsAcute liver failure (ALF), a life-threatening disease characterized by the sudden loss of hepatic function, can occur after an accidental or intentional acetaminophen overdose. MethodsWith the use of an ALF mouse model, we examined both the preventive and therapeutic potential of intravenously administered human umbilical cord–derived mesenchymal stromal cells (hUCMSCs). Primary hUCMSCs were purified from freshly collected full-term umbilical cords and intravenously transplanted into BALB/c mice either before and after ALF induced by acetaminophen intoxication. We found that hUCMSCs significantly improved survival rates and relative liver weight of mice in both pre-ALF and post-ALF animals. Correspondingly, serum levels of markers that reflect hepatic injury (ie, aspartate aminotransferase, alanine aminotransferase and total bilirubin) were significantly attenuated in the group receiving hUCMSC therapy. ResultsMechanistically, we found that the protective potential of intravenously administered hUCMSCs was mediated by paracrine pathways that involved antioxidants (glutathione, superoxide dismutase), the reduction of inflammatory agents (tumor necrosis factor-α, interleukin-6) and elevated serum levels of hepatocyte growth factor. ConclusionsThrough these paracrine effects, intravenously administered hUCMSCs reduced hepatic necrosis/apoptosis and enhanced liver regeneration. Thus, our data demonstrate that intravenously administered hUCMSCs may be useful in the prevention or treatment of acetaminophen-induced ALF.

Osteogenic differentiation of umbilical cord and adipose derived stem cells onto highly porous 45S5 Bioglass®-based scaffolds.

In the context of bone tissue engineering (BTE), combinations of bioactive scaffolds with living cells are investigated to optimally yield functional bone tissue for implantation purposes. Bioactive glasses are a class of highly bioactive, inorganic materials with broad application potential in BTE strategies. The aim of this study was to evaluate bioactive glass (45S5 Bioglass(®)) samples of composition: 45 SiO2, 24.5 CaO, 24.5 Na2O, and 6 P2O5 (in wt%) as scaffold materials for mesenchymal stem cells (MSC). Pore architecture of the scaffolds as well as cell behavior in the three-dimensional environment was evaluated by several methods. Investigations concerned the osteogenic cell attachment, growth and differentiation of adipose tissue derived MSC (adMSC) compared with MSC from human full term umbilical cord tissues (ucMSC) on porous Bioglass(®)-based scaffolds over a cultivation period of 5 weeks. Differences in lineage-specific osteogenic differentiation of adMSC and ucMSC on Bioglass(®) samples were demonstrated. The investigation led to positive results in terms of cell attachment, proliferation, and differentiation of MSC onto Bioglass(®)-based scaffolds confirming the relevance of these matrices for BTE applications.

Poor Vitamin D Status in Healthy Mothers and Infants: Resolution in Neonates on 200 IU Daily

Background and object: According to Institute of Medicine, serum 25-hydroxyvitamin D (S-25-OHD) < 30 nmol/L indicates high risk for rickets. Vitamin D status, as assessed by S-25-OHD, is often poor in normal healthy mothers and their infants in Northern latitudes. Routine supplementation (200 IU/d) of all newborns has recently commenced in Ireland The object of this study was to assess S-25OHD status in maternal and term umbilical cord samples and at follow-up in those neonates with S-25OHD < 30 nmol/L.Methods: S-25-OHD levels were evaluated from maternal (n=44) and umbilical cord samples (n=56) from term normal pregnancies in a consecutive sample of cases. Parents of infants with S-25-OHD < 30nmol/l (n=15) were invited to attend for repeat sampling.Results: 115 samples included 44 maternal and 56 cord samples and 15 follow-up neonatal samples for infants with cord samples < 30nmol/l. The mean of cord blood 25OHD levels were significantly decreased compared with maternal levels. However the neonatal follow-up samples were significantly increased compared to maternal and cord levels in each subgroup (Table1). Table Table Full size table Conclusions: We demonstrated a high prevalence of poor vitamin D status at the time birth in mothers and their infants that was rectified by low dose (200 IU/d) supplementation.

Sequence-Specific Conversion of βA- to βS-Globin by Small Fragment Homologous Replacement In Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 3754 Introduction:An ultimate goal of gene therapy is the development of effective strategies to correct mutant genomic sequences in pathologic cells. To that end, studies have been undertaken to evaluate the therapeutic potential of an oligo/polynucleotide-based sequence-specific gene modification strategy, small fragment homologous replacement (SFHR) in the correction of the mutation giving rise to sickle cell anemia. Small DNA fragments (SDFs) comprising the sickle cell anemia mutation (an A>T transversion in codon 6) and flanking DNA sequences in the human b-globin gene were introduced into Hematopoietic Stem/Progenitor Cells (HSPCs). The studies presented indicated modification at the level of DNA, RNA, and protein when cells were differentiated into erythrocytes. Methods:In this study, SFHR was used to convert A>T in codon 6 of the b-globin gene in CD34+/CD38-/Lin- HSPCs isolated from full term umbilical cord blood as a proof of principle. HSPCs were transfected with a defined number of a 559-bp SDF using the Amaxa electroporation (nucleofection) system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. Erythrocytes were also analyzed using antibodies that differentiate between wild-type hemoglobin A (HBA) and sickle cell hemoglobin S (HBS). Results:RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated conversion of bA- to bS-globin. Sequencing of the 430-bp amplicon showed the A > T conversion. Analysis of the transfected wild-type HSPC-derived erythrocytes with HBA and HBS specific antibodies demonstrated the presence of a subpopulation of cells expressing HBS. These data are consistent with previous studies showing both conversion of bS- to bA-globin in SC1 cells and bA- to bS-globin in HSPCs after electroporation and microinjection of SDF, respectively. Conclusion:These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that they demonstrate long-term SFHR-mediated modification of b-globin following mass transfection by electroporation of HSPCs. Disclosures:No relevant conflicts of interest to declare.

A Retrospective Evaluation of Children Who Have Suffered a Grade III or IV Intraventricular Hemorrhage and Their Parents for Platelet Dysfunction.

AbstractAbstract 1440This study is intended to evaluate children that have survived a grade III or IV intraventricular head bleed and their parents for a potential autosomal dominant inherited platelet dysfunction due to platelet dense granule deficiency (δ-SPD). Intraventricular hemorrhage (IVH) poses a significant risk in premature neonates less than 32 weeks gestation. Infants suffering from high grade (III-IV) periventricular or intraventicular bleeding have mortality rates approaching 50%. In addition, survivors of high grade IVH may suffer severe brain damage or a multitude of other long term neurological sequelae. Thus, we believe that investigation into the pathogenesis of neonatal IVH is not only crucial for the survival of these infants, but also for their quality of life in the future.Current data suggest that the pathogenesis of IVH in the premature infant is related to a loss of ability to regulate cerebral blood flow. Changes in local blood pressure, which would otherwise be autoregulated, remain unrestrained and result in capillary bleeding. Previous prospective studies from our lab have shown that full term neonates have a transient platelet dysfunction at the time of birth (Blood, 100(11), 691a, 2002), which appears to be related to a platelet dense granule storage pool deficiency (δ-SPD). We have also found that 1 day old, full term neonates have mature platelets similar to adults. Our prospective data suggests that at day 3–5 after birth, premature infants of gestational age 26–33 weeks (n=29) have a δ-SPD similar to that observed in full-term umbilical cord blood platelets suggesting a delay in platelet maturation. Only one preemie included in the study had suffered an IVH (grade I); all preemies who suffered Grade III/IV IVH during our study died before we could obtain blood (Blood 108(11):1113, 2006). Parents evaluated had normal platelet storage pools.Our current and on-going retrospective study of children that have survived a grade III/IV IVH and their parents supports our hypothesis that platelet dysfunction may play a significant role in the etiology of IVH. Survivors were identified from a database and parents sent a letter requesting their participation in the study. For those agreeing to participate, informed consent was obtained for this Institutional Review Board approved project. A vial of peripheral blood was obtained from both the IVH surviving child and their parent(s) to determine the platelet dense granule storage pool using the electron microscopy whole mount technique. Parents completed a vetted bleeding questionnaire for assessment of bleeding history/tendencies. Our preliminary data from 6 families reveals that all children surviving a grade III/IV IVH have a δ-SPD with an average of 2.64 ± 0.14 dense granules per platelet (DG/PL) whereas normal = 4–6 DG/PL. Their mothers also have a tendency for δ-SPD (5/6 δ-SPD and 1 within normal limits) with an overall average of 2.93 ± 0.35 DG/PL. Data collected from the questionnaires substantiate the laboratory results with a history of menorrhagia (determined by an established numerical pictorial method) the most significant correlation in 4/6 mothers. The fathers (4/5) tend to have normal numbers of DG/PL (4.06 ± 0.61; 1 had δ-SPD at 1.72 DG/PL). Interestingly, for the one mother with a normal number of DG/PL, the father had δ-SPD. Thus, all children evaluated to date in this study have δ-SPD and a parent who also has δ-SPD. The implication of these preliminary data suggest that potential identification of platelet dysfunction in women at risk for premature delivery could allow for prophylactic intervention for the premature neonate to reduce the morbidity and mortality related to IVH. As this study is currently ongoing, it is anticipated that additional data will be available for presentation. Disclosures:No relevant conflicts of interest to declare.

A Rapid, Simple, and Reproducible Method for the Isolation of Mesenchymal Stromal Cells from Wharton's Jelly Without Enzymatic Treatment

The co-infusion of mesenchymal stromal cells (MSCs) with hematopoietic stem cells could improve the hematopoietic engraftment after cord blood transplant. Adult bone marrow is the major source of MSCs for cell therapy. However, bone marrow aspiration involves an invasive procedure and, in the case of a cord blood transplant, requires the use of a third party. The umbilical cord matrix, called Wharton's jelly (WJ), was previously shown to be a valuable source of MSCs. However, the process of cell separation is not standardized and needs to be optimized. In this study, we focused on the efficiency of the isolation procedure and expansion of cells from WJ MSCs isolated from human full-term umbilical cords. MSCs were isolated from the WJ without enzyme digestion or dissection. The procedure was based only on the plastic adhesion capacities of MSCs. Briefly, umbilical cord segments of 5-10 cm were cut longitudinally and plated with the WJ onto a plastic surface for 5 days in an appropriate culture medium. After removing the cord segment, the culture was pursued until subconfluency. The number of cells and their phenotypes, clonogenic capacities, differentiation capacities, immunomodulation, and hematopoietic supportive functions were evaluated. Using this method, we were able to isolate MSCs from all human umbilical cords analyzed (n = 50). We obtained a mean of 1.4 × 10(8) cells at the second passage and >7 × 10(9) cells at the third. The expanded cells expressed characteristic markers and presented typical functional properties of MSCs such as differentiation capacities, immunologic properties, and hematopoietic supportive functions. In conclusion, we have established a simple, rapid, and reproducible protocol to isolate abundant MSCs from short segments of umbilical cords.

1300 Maternal and Term Umbilical Cord Vitamin D Levels in An Irish Cohort

Background and Object: Vitamin D plays animportant role in skeletal and non skeletal health. We previously reported a high prevalence (about80%) of serum 25-hydroxy-vitamin D (25OHD)insufficiency in preterm VLBW infants. The object ofthis study was to assess 25OHD status in maternaland term umbilical cord samples. Methods: Vitamin D levels were evaluated frommaternal and umbilical cord samples from termnormal pregnancies from January to March 2010and evaluated by radioimmunoasssay. Results: 40 samples included 19 maternal and21 cord samples and 15 maternal and cord pairedsamples. The mean (+/-;Standard deviation) cordblood 25OHD levels were significantly decreasedcompared with maternal levels (29.9+/-;8.3 versus47.9+/-;22.0 nmol/l;p 70nmol/l. Conclusions: We have demonstrated that 80%of preterm VLBW infants have insufficient 25OHDlevels during early postnatal life; but 25% remaininsufficient at follow-up assessment. However inaddition a cohort of normal term mothers and infants have significantly decreased 25OHD levels whichmay be related to the Irish climactic conditions.

Optimization of the culturing conditions of human umbilical cord blood‐derived endothelial colony‐forming cells under xeno‐free conditions applying a transcriptomic approach

Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and characterized by flow cytometry, capillary formation and responsiveness to cytokines. ECFCs were expanded under standard, FBS-containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. ECFC outgrowth in standard medium was successful in 92% of cord blood units. The karyotype of expanded ECFCs remained normal. Without FBS, ECFC initiation and expansion failed. Modest proliferation, changes in cell morphology and organization and cell death have been observed after passaging. Gene ontology analysis revealed a broad down-regulation of genes involved in cell cycle progression and up-regulation of genes involved in stress response and apoptosis. Interestingly, genes participating in lipid biosynthesis were markedly up-regulated. Detection of several endothelial cell-specific marker genes showed the maintenance of the endothelial cell characteristics during serum-free culture. Although ECFCs maintain their endothelial characteristics during serum-free culturing, they could not be expanded. Additional supply of FBS-free media with lipid concentrates might increase the ECFC survival.

Effects of compound musk injection on neurocyte-like differentiation of human umbilical cord-derived mesenchymal stem cells

Objective To observe the effects of compound musk injection on neurocyte-like differentiation of human umbilical cord-derived mesenchymal stem cells (hUMSCs) in vitro. Methods The full-term umbilical cords from healthy neonates were washed with D-Hank' s,and MSCs were separated from them. After flow cytometry was performed,hUMSCs were pre-induced with 10 μg/L basic fibroblast growth factor (bFGF) ,then were induced to neural differentiation by serum-free culture medium containing different concentrations of compound musk injection including 5,10,15,20,25 g/L respectively. The expression of neuron specific enolase (NSE), microtubule-associated protein 2 (MAP-2) and glial fibrillary acid protein (GFAP) was detected by immunocytochemistcy. Results Fluorescence activating cell sorter (FACS) showed that hUMSCs expressed CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD166, HLA-ABC (HLA-I) ,and did not express CD31 ,CD34,CD38,CD45,CD133,HLA-DR (HLA-Ⅱ). After induction for 5 days by DMEM/F12 medium containing different concentrations of compound musk injection, the hUMSCs of each group could differentiate into neuron-like cells. The number of NSE positive cells in 5,10,15,20,25 g/ L groups was 19.00 ±3.61,66.60 ±5.37,60.20 + 10.06,44.80 ±9.44,47. 80 ± 10.45,and that of MAP-2 positive cells was 9.20 ± 3. 27,53.40 ± 10. 92,59.40 ± 10. 41,46. 60 ± 8. 88,46. 80 ± 8. 93, respectively. NSE and MAP-2 positive cells were most plentiful in 10 g/L and 15 g/L groups. But just few of GFAP weak positive cells were detected in all five groups. Conclusion Human umbilical cord is a rich source of MSCs. Adequate concentration of compound musk injection can effectively induce the differentiation of hUMSCs into neural-like cells. Key words: Umbilical cord; Mesenchymal stem cells; Compound musk injection solution; Neuron; Differentiation

Phenotypical and Functional Characterization of Umbilical Cord Blood-Derived Mesenchymal Stromal Cells Expanded in the Presence of Platelet Lysate and Comparison with Their Bone Marrow-Derived Counterpart

The presence of mesenchymal progenitors in full-term umbilical cord blood (UCB) has been object of discussion in recent years, as attempts to obtain these cells have either failed or yielded low frequency of mesenchymal stromal cells (MSCs). MSCs have been so far mainly expanded in vitro in the presence of fetal calf serum (FCS), which potentially carries the risk of both transmitting zoonoses and causing immune reactions against animal proteins. For these reasons, alternative culture supplements, devoid of animal components, such as platelet lysate (PL), have been tested, allowing efficient MSC isolation and expansion from bone marrow (BM). In this study, we tested the ability of PL-additioned medium to isolate and expand ex vivo MSCs from full-term UCB (UCB-MSCs) and we characterized these cells in terms of clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential and biosafety profile, comparing these characteristics with those of PL-expanded BM-MSCs. Moreover, we focused our attention on immunoregulatory properties of UCB-MSCs on alloantigen-specific immune responses and on the mechanisms by which these cells exert their effect. Ten UCB units (median volume 45 ml, range 40–60), from full-term deliveries, were selected according to the following criteria:total nucleated cell (TNC) count ranging from 500 to 750 ×106;isolation performed within 24 hours after delivery;overall cell viability > 75%, investigated by 7-amino-actinomycin D (7-AAD) and Aldeflour (ALDH).Two of the 10 UCB units (20%, UCB3 and UCB6) gave rise to MSC-like clones, which were expanded ex vivo and characterized. UCB-MSCs displayed the typical morphology, immunephenotype and differentiation capacity into osteoblasts and adypocytes reported in the literature. Although displaying a rather low clonogenic efficiency, UCB-MSCs showed to have a higher proliferative potential compared to BM-MSCs, as demonstrated by the calculated cumulative cell counts from P0 to P5. Thereafter, UCB3- and UCB6-MSCs displayed a progressive decrease in proliferative capacity, until they reached senescence after 83 (P10) and 90 (P11) days of culture. The cells progressively died during the senescence period, without showing any alteration in morphology or proliferative rate. The lack of spontaneous transformation into tumor cells was demonstrated by both the absence of telomerase activity and hTERT transcripts and by molecular karyotyping through array-Comparative Genomic Hybridization (array-CGH) assay. The immune-regulatory effect of UCB-MSCs on alloantigen-specific immune response in mixed lymphocyte culture (MLC) was investigated, together with some of the mechanisms potentially responsible for this effect, including PGE2 production and IDO activity. We found that, similarly to BM-MSCs, UCB-MSCs expanded in PL are able to:strongly inhibit alloantigen-induced lymphocyte subset (CD3+, CD4+, CD8, CD3negCD56+ NK lymphocytes) proliferation;decrease alloantigen-induced cytotoxic activity;increase secretion of IL-6 and IL-10 in MLC supernatant.While the addition of BM-MSCs to MLC increased the percentage of CD4+CD25+FoxP3+T cells, the addition of UCB-MSCs did not result in any increase of this cell subset. Moreover, we found that the immune modulation of UCB-MSCs is apparently due to PGE2 production, while the addition of IDO-specific inhibitor was not able to reverse the suppressive effect exerted by MSCs. Altogether, these data indicate that relevant differences exist between UCB- and BM-MSCs, ex vivo cultured in the presence of PL, in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties. These aspects may be relevant for the clinical application of UCB-MSCs.

Isolation and characterisation of mesenchymal stem cells in Wharton's jelly of the human umbilical cord: potent cells for cell-based therapies in paediatric surgery?

BACKGROUND: Mesenchymal stem cells (MSC) are known to have the ability to differentiate into various lineages of mesenchymal tissue. They are widely distributed in a variety of tissues in the adult human body and are also present in the foetal environment. However, MSC do only attribute for a rare population in these tissues. In this study we have evaluated the possibility of MSC or cells with MSC-like potency being present in the umbilical cord (UC). METHODS: Term UC was collected and stored in sterile, saline solution. Upon preparation, the UC was cut into segments of 1 cm length, the vessels were striped manually and the tissue immersed in an enzyme cocktail for 3 h at 37 °C. Isolated umbilical cord mesenchymal stem cells (UCMSC) were extracted by low-speed centrifugation, suspended and cultured. RESULTS: (1) It was possible to isolate UCMSC in sufficient quantities and (2) the cells could be cultured easily. (3) The cells obtained from our experiments demonstrated a fibroblast-like phenotype. (4) They could expand when cultured and induced to differentiate into several different types of cells. (5) The cells were found to express mesenchymal markers (CD13, CD105) but not haematopoetic lineage markers (CD14 and CD34). CONCLUSIONS: Our observations confirmed that MSC are present in the human UC in sufficient quantity for isolation. This tissue should be considered a valuable resource for cell-based therapies, especially in paediatric surgery.