Fraction Of Umbilical Cord Blood Research Articles

Racial differences in IGF1 methylation and birth weight

BackgroundThe birth weight of Black neonates in the United States is consistently smaller than that of their White counterparts. Epigenetic differences between the races may be involved in such disparities. The goal of these analyses was to model the role of IGF1 methylation in mediating the association between race and birth weight. Data was collected on a cohort of 87 live born infants. IGF1 methylation was measured in DNA isolated from the mononuclear fraction of umbilical cord blood collected after delivery. Quantitative, loci-specific methylation was assessed using the Infinium HumanMethylation27 BeadArray (Illumina Inc., San Diego, CA). Locus specific methylation of the IGF1 CpG site was validated on a subset of the original sample (N = 61) using pyrosequencing. Multiple linear regression was used to examine relationships between IGF1 methylation, race, and birth weight. A formal mediation analysis was then used to estimate the relationship of IGF1 methylation to race and birth weight.ResultsBlack race was associated with a 7.45% decrease in gestational age-adjusted birth weight (aBW) (P = 0.04) and Black infants had significantly higher IGF1 methylation than non-Black infants (P < 0.05). A one standard deviation increase in IGF1 methylation was associated with a 3.32% decrease in aBW (P = 0.02). Including IGF1 methylation as a covariate, the effect of Black race on aBW was attenuated. A formal mediation analysis showed that the controlled direct effect of Black race on aBW was −6.26% (95% CI = −14.15, 1.06); the total effect of Black race on IGF1 methylation was −8.12% (95% CI = −16.08, −0.55); and the natural indirect effect of Black race on aBW through IGF1 methylation was −1.86% (95% CI = −5.22, 0.18)ConclusionThe results of the mediation analysis along with the multivariable regression analyses suggest that IGF1 methylation may partially mediate the relationship between Black race and aBW. Such epigenetic differences may be involved in racial disparities observed in perinatal outcomes.

Conditioned Medium Originated From Lethally Irradiated Umbilical Cord Blood-Derived Mononuclear Cells Has Different Pro-Angiogenic Effects Over Mature and Progenitor Endothelial Cells In Vitro

IntroductionIschemic cardiovascular diseases are a leading cause of mortality in the industrialized world. Cell-based therapies have been tested to promote revascularization, mainly through the release and actions of paracrine factors that may prevent apoptosis and support migration and proliferation of resident endothelial cells (EC) and circulating endothelial progenitors (EPC). The mononuclear cell fraction of umbilical cord blood (CB-MNC) is rich in pluripotent progenitors and represents an attractive cell source for therapeutic angiogenesis. In order to demonstrate the paracrine action of CB-MNC we tested the angiogenic potential of conditioned medium (CM) derived from apoptotic CB-MNC evaluating the effects exerted over mature EC and peripheral blood (PB)-derived EPC in terms of proliferation, migration and capillary-network formation in vitro. MethodsHuman Umbilical Vein Endothelial Cells (HUVEC, Lonza) were cultured in Endothelial Basal Medium (EBM2) supplemented with SingleQuotes (EGM2). EPCs were obtained from PB as “Endothelial Colony Forming Cells” (ECFC). Both HUVEC and ECFC were used for subsequent experiments from passage 2 to passage 6.MNC were isolated from CB by density gradient centrifugation, gamma-irradiated (70Gy) to induce apoptosis and incubated in EBM2 for 72h to obtain the release of CM. CM was recovered from culture, centrifuged (4000 rpm 10 min) to remove cell debris and stored at -80°C. MNC viability after gamma-irradiation was evaluated by flow cytometry (7-AAd staining). RayBio Human Angiogenesis Array and ELISA assays (to quantify PDGF-AB, VEGF165, EGF, IGF-I) were used to assess CM soluble factors content.The angiogenic potential of soluble factors released by gamma-irradiated MNC was assessed by Matrigel capillary-like network formation assay using the Transwell Permeable Supports and confirmed using the sole CM in the Matrigel assay. CM capacity to support HUVEC and ECFC viability was assessed by the MTT assay and the capacity to induce HUVEC and ECFC migration by the Chemicon Cell Migration Assay. EBM2 was used as control medium. All experiments were repeated at least 4 times. ResultsGamma-irradiation induced a significant time-dependent reduction in MNC viability (p<0.05), reaching 41±7% at 72h. The difference in viability decrease between irradiated and not irradiated MNC was statistically significant over time (p<0.001).CM contained various cytokines and growth factors involved in the regulation of angiogenesis, wound healing, extracellular matrix remodeling and inflammation as showed by Angiogenesis Array. ELISA assays showed that irradiated MNC released an increasing amount of PDGF-AB, VEGF165 and EGF over time, while IGF-I was not detected in the CM.Soluble factors released from irradiated MNC in the Transwell system showed a significant pro-angiogenic effect over HUVEC (p<0.001, Figure 1a) as well as over ECFC (p<0.001, Figure 1b). [Display omitted] Results obtained with the Matrigel Transwell system using irradiated MNC were confirmed employing CM from irradiated MNC cultured 72 hours: CM induced capillary-like network formation in both HUVEC (p<0.001, Figure 1c) and ECFC (p<0.01, Figure 1d).CM compared to control medium significantly increased HUVEC viability (p<0.05, Figure 2a) but not ECFC viability (Figure 2b) and significantly enhanced migration of both HUVEC (p<0.001, Figure 3a) and ECFC (p<0.001, Figure 3b). [Display omitted] [Display omitted] ConclusionOur experiments show that soluble factors released by lethally irradiated CB-MNC have an important angiogenic effect, inducing HUVEC but not ECFC proliferation, HUVEC and ECFC migration and capillary network formation in vitro. These results outline an interesting difference between mature and progenitor EC in their response to CM, which may reflect the different physiological roles that mature and progenitor EC exert during angiogenesis.This study confirms the paracrine effect of factors released from stem cells contained in the MNC fraction, which could be exploited in regenerative medicine to induce therapeutic revascularization using CB, an easy available stem cells source. Disclosures:No relevant conflicts of interest to declare.

Adipose stem cell-derived osteoblasts sustain the functionality of endothelial progenitors from the mononuclear fraction of umbilical cord blood

Vascularization is the most pressing issue in tissue engineering (TE) since ensuring that engineered constructs are adequately perfused after in vivo transplantation is essential for the construct’s survival. The combination of endothelial cells with current TE strategies seems the most promising approach but doubts persist as to which type of endothelial cells to use. Umbilical cord blood (UCB) cells have been suggested as a possible source of endothelial progenitors. Osteoblasts obtained from human adipose-derived stem cells (hASCs) were co-cultured with the mononuclear fraction of human UCB for 7 and 21days on carrageenan membranes. The expression of vWF and CD31, and the DiI-AcLDL uptake ability allowed detection of the presence of endothelial and monocytic lineages cells in the co-culture for all culture times. In addition, the molecular expression of CD31 and VE-cadherin increased after 21days of co-culture. The functionality of the system was assessed after transplantation in nude mice. Although an inflammatory response developed, blood vessels with cells positive for human CD31 were detected around the membranes. Furthermore, the number of blood vessels in the vicinity of the implants increased when cells from the mononuclear fraction of UCB were present in the transplants compared to transplants with only hASC-derived osteoblasts. These results show how endothelial progenitors present in the mononuclear fraction of UCB can be sustained by hASC-derived osteoblast co-culture and contribute to angiogenesis even in an in vivo setting of inflammatory response.

Umbilical Cord Blood Research: Current and Future Perspectives

Umbilical cord blood (UCB) banking has become a new obstetrical trend. It offers expectant parents a biological insurance policy that can be used in the event of a child or family member's life-threatening illness and puts patients in a position of control over their own treatment options. However, its graduation to conventional therapy in the clinical realm relies on breakthrough research that will prove its efficacy for a range of ailments. Expanding the multipotent cells found within the mononuclear fraction of UCB so that adequate dosing can be achieved, effectively expanding desired cells ex vivo, establishing its safety and limitations in HLA-mismatched recipients, defining its mechanisms of action, and proving its utility in a wide variety of both rare and common illnesses and diseases are a few of the challenges left to tackle. Nevertheless, the field is moving fast and new UCB-based therapies are on the horizon.

Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however, other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets, purified ALDH(br) CD34+, ALDH(neg) CD34+, and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison, ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development.

Stroma from different developmental stages of fetal liver has different effects on human mononuclear fraction of umbilical cord blood during ex vivo co-culture

Fetal liver (FL) has transient and intense hematopoietic activity during gestation. It has been shown that hematopoietic activity in FL begins at embryonic day (E)11, is maximal at E16 and rapidly ceases thereafter. This development of FL is simultaneous with transition of several hematopoietic features. Stromal cells (SCs) appear to regulate hematopoiesis in FL. To compare in vitro hematopoietic support ability of FL SCs from different developmental stages, we established three mouse primary FL SCs from different gestation days and compared their capacity in supporting the growth and differentiation of human umbilical cord blood, by flow cytometry and long-term culture-initiating cell (LTC-IC) assays. Results showed that expansion of hematopoietic progenitor in an E15.5 FL SC microenvironment was superior to that in E12.5 and E18.5 FL SC microenvironments. Co-culture on E15.5 FL SCs resulted in a 3.04-fold increase in early CD34+/CD38− progenitors and a 6.4-fold increase in LTC-ICs after 2 weeks. However, the comparison of CD2+/CD19− cell output and CD19+ cell output in different conditions did not attain statistical significance. These results show that murine E15.5 FL SCs are most effective in sustaining stem cell function along with greatest expansion of total nuclear cells of human umbilical cord blood (hUCB). We conclude that the change in SC function is responsible for the transition of hematopoietic function in FL.

Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers.

The stromal cell population in bone marrow has been the focus of much attention since it has been shown that this cell population can be expanded and differentiated into cells with the phenotype of bone, cartilage, muscle, stroma, neural, and fat cells. We evaluated umbilical cord blood (UCB) for the presence of these cells. From the mononuclear fraction of UCB, we demonstrated the presence of a subset of cells that have been maintained in continuous culture for more than 6 months (>10 passages). These adherent cell populations express adhesion molecules CD13+, CD29+, and CD44+, but not antigens of hematopoietic differentiation. Exposure of these cells to osteogenic agents resulted in an increase in expression of alkaline phosphatase and the appearance of hydroxyapatite nodules by Von Kossa staining. Incubation with adipogenic agents resulted in morphological change and staining with Oil Red O. In addition, when exposed to basic fibroblast growth factor and human epidermal growth factor the cells underwent changes consistent with cells of neural origin. These changes were demonstrated by a combination of immunofluorescent labeling and Western immunoblots for neural-specific markers. Thus, similar to what has been previously reported with bone marrow, cord blood contains a population of cells that can be expanded in culture and are able to express the phenotype of multiple lineages. Cord blood multilineage cells are slower to establish in culture, have a lower precursor frequency and a lower level of bone antigen expression, and lack constitutive expression of neural antigens when compared to bone marrow, suggesting a more primitive population. Cord blood may prove to be a new source of cells for cellular therapeutics for stromal, bone, and, potentially, neural repair.

Quantitative Analysis of the Bidirectional Fetomaternal Transfer of Nucleated Cells and Plasma DNA

Recently, much interest has been generated on the fetomaternal transfer of nucleated cells and plasma DNA. However, there has been no systematic quantitative comparison of these two directions and two modalities of trafficking within the same study population. The fetus-to-mother transfer of nucleated cells and plasma DNA in pregnant women carrying male babies was studied using a real-time quantitative PCR assay for the S:RY gene. For mother-to-fetus transfer, real-time quantitative PCR assays for the insertion/deletion polymorphisms involving the glutathione S:-transferase M1 and angiotensin-converting enzyme genes were used. Of the 50 informative mother-baby pairs, maternal DNA was detected in the cellular fraction of umbilical cord blood in 24% of cases (12 of 50), at a median fractional concentration of 2.6 x 10(-4) (interquartile range, 1.7 x 10(-4) to 3.6 x 10(-4)). In the plasma fraction of cord blood, maternal DNA was detected in 30% (15 of 50) of cases at a median fractional concentration of 3 x 10(-3) (interquartile range, 1 x 10(-3) to 1.6 x 10(-2)). For the other direction of trafficking, fetus-to-mother transfer of nucleated cells was detected in 26% of cases (13 of 50) at a median fractional concentration of 3.2 x 10(-4) (interquartile range, 0.6 x 10(-4) to 7.6 x 10(-4)). In the plasma fraction, fetal DNA was detected in 100% of maternal plasma (50 of 50) at a median fractional concentration of 3 x 10(-2) (interquartile range, 1.4 x 10(-2) to 5. 3 x 10(-2)). This study indicated that significantly more fetal DNA is present in the plasma of pregnant women compared with DNA from the cellular fraction of maternal blood. In addition, maternal DNA was demonstrated in both the cellular and plasma fractions of cord blood after delivery. This study has therefore determined the fundamental quantitative values for the bidirectional fetomaternal cellular and plasma DNA traffic.

Methods of isolation and cryopreservation of stem cells from cord blood.

Successful reconstitution of the hematopoietic system by umbilical cord blood (UCB) stem cells has sparked interest in large-scale banking of UCB. This would only be feasible if UCB samples could be frozen in small volumes. Early efforts to fractionate UCB produced significant losses of stem and progenitor cells. Several groups have proposed techniques to concentrate the stem/progenitor cell fraction of UCB, with good recovery. These are described, together with the principles of cryopreservation of stem cells. It seems feasible to store UCB samples on a large scale, although the different fractionation methods need to be compared, and the optimal, most efficient technique must be determined.