Human Cord Serum Research Articles

The α-Fetoprotein Molecule has One or Two Rigid Domains Depending on the Protein Purification Procedure

Abstract: α-Fetoprotein samples isolated from human cord serum by different methods were compared using circular dichroism, scanning microcalorimetry and fluorescence spectroscopy. Their structural properties and conformational stability were appreciably discriminated. In particular, the protein molecule had either one or two relatively rigid domains which can be co-operatively disrupted by heating or urea treatment. This is due to the difference of the isolation procedures in their capabilities to strip the natural ligands from the protein molecule.

Human umbilical cord serum as an alternative to fetal bovine serum for in vitro expansion of umbilical cord mesenchymal stromal cells.

In the use of bovine fetal serum (FBS) there is concern about the possibility of disease transmission from animal to human. Therefore, it seems necessary to create culture conditions free of animal serum, especially in cell therapy. The aim of this study was to evaluate the feasibility of replacing human umbilical cord serum (hUCS) with FBS for in vitro expansion of umbilical cord mesenchymal stromal/stem cells (UC-MSCs). Here, UC-MSCs were cultured for five days in media supplemented either by hUCS or commercial FBS (Gibco and HyClone) to compare their viability, proliferation, morphology, Immunophenotype and differentiation potential. Our data shows that use of 5% and/or 10% hUCS, resulted in a tenfold increase in the number of MSCs; While in the presence of commercial FBS, this figure reached a maximum of five times. Notably, the rate of cell proliferation in the group containing 2% hUCS was the same as the groups containing 10% commercial FBS. Furthermore, there was no significant difference between groups in terms of viability, surface markers, and multilineage differentiation potential. These results demonstrated that hUCS can efficiently replace FBS for the routine culture of MSCs and can be used ideally in manufacturing process of UC-MSCs in cell therapy industry.

Tubulin-binding peptide RR-171 derived from human umbilical cord serum displays antitumor activity against hepatocellular carcinoma via inducing apoptosis and activating the NF-kappa B pathway.

ObjectivesHepatocellular carcinoma (HCC) still presents a high incidence of malignant tumours with poor prognosis. There is an urgent need for new therapeutic agents with high specificity, low toxicity and favourable solubility for the clinical treatment of HCC.Materials and MethodsThe bioactivity of human umbilical cord serum was investigated by proteomics biotechnology and a primitive peptide with certain biological activity was identified. The antitumour effect of RR‐171 was detected by cell viability assay in vitro, and determined by subcutaneous xenograft models assay and miniPDX assay in vivo. Pull‐down experiments were conducted to identify the potential targeting proteins of RR‐171. Immunofluorescence assay and tubulin polymerization assay were conducted to explore the relationship between RR‐171 and α‐tubulin. Fluorescence imaging in xenograft models was used to explore the biodistribution of RR‐171 in vivo. A phosphospecific protein microarray was performed to uncover the underlying signalling pathway by which RR‐171 induces tumour cell death.ResultsThe results indicated that RR‐171 could be effective in the treatment of HCC in vivo and in vitro. RR‐171 could aggregate significantly in solid tumours and had no obvious systemic toxicity in vivo. RR‐171 could interact with α‐tubulin and activate the NF‐Kappa B pathway in HCC cells.ConclusionsTaken together, RR‐171 exhibited significant antitumour activity against HCC in vivo and in vitro and could potentially be used in the clinical application of HCC.

Proteomic analysis of human umbilical cord serum exosomes using mass spectrometry and preliminary study of their biological activities in liver cancer cell lines.

Exosomes are membranous extracellular vesicles 50-100 nm in size, which are involved in cellular communication via the delivery of proteins, lipids and RNA. Emerging evidence shows that exosomes play a critical role in cancer. It has recently been revealed that maternal and umbilical cord serum (UCS)-derived exosomes may enhance endothelial cell proliferation and migration. However, the role of exosomes isolated from the human umbilical cord in cancer development has not been investigated. To explore the potential differences in the composition and function of proteins from umbilical serum exosomes (UEs) and maternal serum exosomes, a proteomic analysis of exosomes was conducted using mass spectrometry and bioinformatics. Moreover, Cell Counting Kit-8 assays and flow cytometry were used to study the biological effects of UEs on liver cancer cell lines. The present study demonstrated that UCS was enriched with proteins involved in extracellular matrix-receptor interactions, which may be closely related to cell metastasis and proliferation. The findings further indicated that exosomes derived from human umbilical serum could inhibit the viability and induce apoptosis of liver cancer cells. This suggests that UCS-derived exosomes may represent potential leads for the development of biotherapy for liver cancer.

Bioprinted Collagen-Based Cell-Laden Scaffold With Growth Factors for Tympanic Membrane Regeneration in Chronic Perforation Model.

With the recent development of bioprinting technology, various attempts have been made to replace bioprinting technologies and regenerative medicine are more directed towards transplantation/reconstructive surgeries only with the implantation of scaffolds. The purpose of this study is to determine whether the growth factors, human umbilical cord serum (hUCS) and bFGF (basic fibroblast growth factor), have a synergistic effect on eardrum regeneration, when used with a cell-printed scaffold in a chronic tympanic membrane perforation (TMP) model. In this study, in vitro cellular activities for bioprinted cell-laden collagen scaffolds using human adipose stem cells (hASCs) and supplemented with 10 [Formula: see text]/mL hUCS and 10 ng/mL bFGF were performed. The mixture of the growth factors in the cell-laden structures effectively affects various in vitro cellular responses including the proliferation of hASCs and the migration of keratinocytes due to the synergistic effect of the growth factors and hASCs. For the in vivo evaluation, a rat TMP model was used, and the TMP regeneration was assessed by otoscopic examination, hearing threshold measurement, and histologic examination. Although the cell-laden structure containing hUCS was more enhancing effect compared to the structure with bFGF, more synergistic effect in the structure using hUCS/bFGF was observed. Based on the results, we believe that the cell-laden structure incorporating hUCS and bFGF can induce significant regeneration of chronic tympanic membrane perforation.

Effects of fibrous collagen/CDHA/hUCS biocomposites on bone tissue regeneration

Collagen- and bioceramic-based composites have been widely used in hard tissue engineering because they are analogous to the organic/inorganic constituents of native bones. However, biocomposites based on collagen and bioceramics show low mechanical stiffness and limited osteogenic activities. To elevate the low biophysical and biological activities, we have introduced a new biocomposite structure. Herein, we propose a biocomposite mimicking not only the physical structure of the extracellular matrix (ECM) structure but also the biochemical components of native bone tissues. Several components including fibrillated collagen, calcium-deficient hydroxyapatite (CDHA) obtained from α-tricalcium phosphate hydrolysis, and human umbilical cord serum (hUCS) were used to generate a unique structure of the biocomposite. The 3D-printed composites were topographically similar to the nanofibrous ECM and exhibited a mechanically stable structure. We also evaluated the in vitro biocompatibilities of the biocomposite using human adipose stem cells and found that the collagen/hUCS/CDHA scaffold accelerated the in vitro osteogenic differentiation of human adipose-derived stem cells and in vivo osteogenesis in a mastoid obliterated rat model.

Use of Human Umbilical Cord Serum to Treat Animal Skin Burns

Objective: The objective of the study was to test the hypothesis, that umbilical cord blood crude serum applied topically with its high concentration of growth factors may promote an early healing for animal models with burn injury.
 Methods: Fresh human umbilical cord blood UCBS was collected and screened for transmitted diseases such as hepatitis B, hepatitis C and HIV. Mice and rabbits were scalded by boiling water and chemical burning by NaOH, then treated with UCBS. In comparison with a Cetrimide treated group and a control group (without treatment). The UCBS was subjected to microbial testing to demonstrate the presence or absence of extraneous viable contaminating microorganisms.
 Results: Topical application of UCBS promoted the healing process; complete healing was seen after 10 days for the mice group and in 7 days in the rabbit group. Cetrimide group applied to the rabbit showed slow healing and needed 10 days for hair regrowth. Control group in both mice and rabbits showed very slow response and the burn area diameter remained the same for over 10 days, and no hair regrowth was obvious after 10 days. 
 Conclusion: The results of the current study indicated that UCBS is a promising therapy for the healing of burns by boiling water and/or chemical substance, more clinical trials are needed to further explore the long-term effects after UCBS use.

Therapeutic effects of three human-derived materials in a mouse corneal alkali burn model*

Purpose: To compare the therapeutic effects of human derivatives in a mouse alkali burn model.Methods: The right eyes of mice were injured using NaOH. After alkali injury, one of the following agents was topically administered for 7 d: human amniotic membrane (hAM) suspension, human umbilical cord serum (hUCS), and human peripheral blood serum (hPBS), or saline. The epithelial defect areas on days 1, 2, and 3 degrees of opacity on days 2, 3, and 7, and corneal neovascularization (NV) areas on day 7 were evaluated. Histologic examination and mRNA expression levels of tumour necrosis factor (TNF)-α, interleukin (IL)-6, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 were also evaluated on day 7.Results: The epithelial defect areas in the hUCS group were smaller than those in the control and hPBS groups on day 3 (p < .05, respectively). The epithelial defect areas in the hAM suspension group showed smaller than those in the control and hPBS groups on days 1 and 2 (p < .05, respectively). The degrees of opacity were lower in all treatment groups than that of the saline control group on day 7 (p < .05, respectively). Corneal NV areas were not different among groups on day 7 (p = 0.20). The expression levels of TNF-α, IL-6, MMP-8, and MMP-9 mRNA and the infiltration of the inflammatory cells in all treatment groups were lesser than those in the control group on day 7 (p< .05, respectively).Conclusions: All treatments reduced inflammatory reactions and corneal opacity development. Corneal reepithelialization was faster in the hUCS group.

TBBPA and Its Alternatives Disturb the Early Stages of Neural Development by Interfering with the NOTCH and WNT Pathways.

Tetrabromobisphenol A (TBBPA), as well as its alternatives Tetrabromobisphenol S (TBBPS) and Tetrachlorobisphenol A (TCBPA), are widely used halogenated flame retardants. Their high detection rates in human breast milk and umbilical cord serum have raised wide concerns about their adverse effects on human fetal development. In this study, we evaluated the cytotoxicity and neural developmental toxicity of TBBPA, TBBPS, and TCBPA with a mouse embryonic stem cell (mESC) system, at human body fluid and environmental relevant doses. All the three compounds showed similar trends in their cytotoxic effects. However, while TBBPA and TBBPS stimulated ESC neural differentiation, TCBPA significantly inhibited neurogenesis. Mechanistically, we demonstrated that, as far as the NOTCH (positive regulator) and WNT (negative regulator) pathways were concerned, TBBPA only partially and slightly disturbed them, whereas TBBPS significantly inhibited the WNT pathway, and TCBPA down-regulated the expression of NOTCH effectors but increased the WNT signaling, actions which both inhibited neural specification. In conclusion, our findings suggest that TBBPS and TCBPA may not be safe alternatives to TBBPA, and their toxicity need to be comprehensively evaluated.

The Tissue Culture Laboratory of Dr. George Otto Gey 60yrs ago as recalled by a former student.

George Otto Gey was a pioneer in tissue culture, having introduced the roller drum, the HeLa cell line, and the use of human fetal cord serum and beef embryo extract. During his career (1920s-1960s), the field of tissue culture was in its infancy and not yet dependent upon commercial biological supply houses. While the early techniques of cell culture have been greatly improved upon, of historical interest may be personal observations of the Gey Tissue Culture Laboratory, Johns Hopkins Medical School, as recalled by a medical student working there in the 1950s. Dr. Gey served as a founding member and executive of the Tissue Culture Commission (TCC) and became the first president of the Tissue Culture Association (TCA).

Successful derivation of xeno-free mesenchymal stem cell lines from endometrium of infertile women

Transplantation of mesenchymal stem cells (MSC) can effectively repair endometrial deficiencies, including infertile patients with a problem of inadequate endometrium thickness. Although, MSC derived from different organ sources have a similarity of MSC specific characteristics, endometrial stem cells (EMSC) are temporally regulated throughout the menstrual cycle in a micro-environmental niche found only in endometrial tissue. Given the micro-environment niche, developing treatments for endometrial disorders with EMSC should be a top priority. To provide EMSC that afford safety for therapeutic usage, we have established a completely xeno-free EMSC line derivation protocol using human allogenic umbilical cord serum instead of animal derived reagents, and proved that it is feasible to generate xeno-free EMSC lines from infertile patient donors using these conditions. Our results demonstrate the successful derivation of xeno-free EMSC lines from 10 out of 10 infertile patients. The resultant xeno-free EMSC lines showed typical MSC morphology, phenotypic markers, differentiation capacity, telomere length and normal karyotypes. They showed superior proliferation capability, but lower expression of proto-oncogenes, to the lines generated under standard (animal derived reagents) culture. Biosafety of xeno-free EMSC lines also displayed in retention of immunosuppressive ability, epigenetic stability by imprinted genes expression, proto-oncogenes expression and no mutation of specific codon on p53 tumor suppressor gene. Taken together, these data indicate that our cells may be safe for clinical use. In conclusion, we have succeeded in establishing completely xeno-free EMSC lines and demonstrate for the first time that autogenic and xeno-free EMSC lines can be generated from infertile women.

Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum

Fetal bovine serum (FBS) is widely used in cell culture laboratories, risk of zoonotic infections and allergic side effects create obstacles for its use in clinical trials. Therefore, an alternative supplement with proper inherent growth-promoting activities is demanded. To find FBS substitute, we tested human umbilical cord blood serum (hUCS) for proliferation of human umbilical cord matrix derived mesenchymal stem cells (hUC-MSCs) and human bone marrow-derived mesenchymal cells (hBM-MSCs). Umbilical cord blood of healthy neonates, delivered by Caesarian section, was collected and the serum was separated. hUC-MSCs and hBM-MSCs were isolated and characterized by assessment of cell surface antigens by flow cytometry, alkaline phosphatase activity and osteogenic/adipogenic differentiation potential. The cells were then cultured in Iscove's Modified Dulbecco's Medium (IMDM) by conventional methods in three preparations: 1- with hUCS, 2- with FBS, and 3- without serum supplements. Cell proliferation was measured using WST-1 assay, and cell viability was assessed by trypan blue staining. The cells cultured in hUCS and FBS exhibited similar morphology and mesenchymal stem cells properties. WST-1 proliferation assay data showed no significant difference between the proliferation rate of either cells following hUCS and FBS supplementation. Trypan blue exclusion dye test also revealed no significant difference for viability between hUCS and FBS groups. A significant difference was detected between the proliferation rate of stem cells cultured in serum-supplemented medium compared with serum-free medium. Our results indicate that human umbilical cord serum can effectively support proliferation of hBM-MSCS and hUC-MSCs in vitro and can be used as an appropriate substitute for FBS, especially in clinical studies.

Effect of polycaprolactone/collagen/hUCS microfiber nerve conduit on facial nerve regeneration

Nerve conduits have been used to bridge nerve gaps without the donor site morbidity associated with nerve grafting. To fabricate artificial nerve conduits, various biological, synthetic, and natural materials have been applied. In this study, we suggest a new fibrous nerve conduit consisting of PCL microfibers supplemented with collagen and human umbilical cord serum (hUCS). The proposed PCL-based nerve conduit exhibited significantly higher bioactivities in vitro and in vivo, compared to pure PCL and PCL/collagen fibrous conduits. Based on these results, we believe that the PCL/collagen/hUCS fibrous conduit provides more favorable micro-environmental conditions for facial nerve (FN) regeneration and has more therapeutic potential for the regeneration of FN transectional damage than the autograft technique.

The effect of ovine, bovine and human umbilical cord blood sera on in vitro maturation of sheep oocytes

The aim of the present study was to evaluate the effect of ovine umbilical cord serum (OUCS), bovine umbilical cord serum (BUCS) and human umbilical cord serum (HUCS) on in vitro maturation, cleavage and developmental capacity of sheep oocytes. Oocytes were treated in maturation media with different concentration (10 and 20%) of OUCS, BUCD, HUCS and FCS to examine effects on early embryonic development. The processing results showed that the addition of 10% OUCS (27.1%) and BUCS (24.8%) can improve (P<0.05) the mean blastocyst formation compared maturation medium with FCS (17.1%). However, there was no significant difference (P>0.05) in blastocyst rate between OUCS (20.2%), BUCS (21.8%), HUCS (18.2%) and FCS (23.2%) when sera concentrations were increased from 10 to 20%. In conclusion, OUCS and BUCS alone (without growth factor supplement) could improve the embryo production and early embryonic development of sheep oocytes. However, the positive effects of umbilical cord sera on embryonic development may remove by high concentrations of umbilical cord sera.

A polycaprolactone/silk-fibroin nanofibrous composite combined with human umbilical cord serum for subacute tympanic membrane perforation; an in vitro and in vivo study.

Due to its good mechanical reinforcement and large number of basic amino acids, silk fibroin (SF) has been widely used in tissue regenerative applications as a bioactive dispersed phase of a biocomposite system. In this work, we fabricated an electrospun polycaprolactone (PCL)/SF composite and combined the composite scaffold with human umbilical cord serum (UCS) to facilitate cell growth. Various in vitro cellular activities (cell-viability, MTT assay, and fluorescence analysis) for the electrospun pure PCL, pure SF, PCL/SF, and PCL/SF/UCS scaffolds were compared. The PCL/SF/UCS based scaffolds showed highly improved bioactive properties compared with pure PCL and SF. The efficiency and feasibility for regenerating in vivo subacute tympanic membrane (TM) perforation in guinea pigs was also evaluated for the PCL/SF/UCS scaffold. The closure of the TM perforation using the scaffold was rapid; the auditory brainstem response (ABR) threshold at all frequencies of the composite demonstrated significant recovery, approaching normal levels.

Regeneration of chronic tympanic membrane perforation using 3D collagen with topical umbilical cord serum

Chronic tympanic membrane (TM) perforation is one of the most common otology complications. Current surgical management of TM perforation includes myringoplasty and tympanoplasty. The purpose of this study was to evaluate the efficacy and feasibility of three dimensional (3D) porous collagen scaffolds with topically applied human umbilical cord serum (UCS) for the regeneration of chronic TM perforation in guinea pigs. To achieve this goal, we fabricated porous 3D collagen scaffolds (avg. strut diameter of 236±51μm, avg. pore size of 382±67μm, and a porosity of 96%) by using a 3 axis robot dispensing and low temperature plate systems. Guinea pigs were used in a model of chronic TM perforation. In the experimental group (n=10), 3D collagen scaffold was placed on the perforation and topically applied of UCS every other day for a period of 8 days. The control group ears (n=10) were treated with paper discs and phosphate buffered saline (PBS) only using the same regimen. Healing time, acoustic-mechanical properties, and morphological analysis were performed by otoendoscopy, auditory brainstem response (ABR), single-point laser Doppler vibrometer (LDV), optical coherence tomography (OCT), and light microscopic evaluation. The closure of the TM perforation was achieved in 100% of the experimental group vs. 43% of the control group, and this difference was statistically significant (p=0.034). The ABR threshold at all frequencies of the experimental group was significantly recovered to the normal level compared to the control group. TM vibration velocity in the experimental group recovered similar to the normal control level. The difference is very small and they are not statistically significant below 1kHz (p=0.074). By OCT and light microscopic examination, regenerated TM of the experimental group showed thickened fibrous and mucosal layer. In contrast, the control group showed absence of fibrous layer like a dimeric TM.

Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections.

A single neonatal exposure to perfluorohexane sulfonate (PFHxS) affects the levels of important neuroproteins in the developing mouse brain

Perfluorohexane sulfonate (PFHxS) is an industrial chemical and belongs to the group of perfluorinated compounds (PFCs). It has recently been shown to cause developmental neurobehavioral defects in mammals. These compounds are commonly used in products such as surfactant and protective coating due to their ability to repel water- and oil stains. PFCs are globally found in the environment as well as in human umbilical cord blood, serum and breast milk. In a previous study on other well-known PFCs, i.e. PFOS and PFOA, it was shown that neonatal exposure caused altered neuroprotein levels in the hippocampus and cerebral cortex in neonatal male mice. The present study show that neonatal exposure to PFHxS, during the peak of the brain growth spurt, can alter neuroprotein levels, e.g. CaMKII, GAP-43, synaptophysin and tau, which are essential for normal brain development in mice. This was measured for both males and females, in hippocampus and cerebral cortex. The results suggest that PFHxS may act as a developmental neurotoxicant and the effects are similar to that of PFOS and PFOA, but also to other substances such as PCBs, PBDEs and bisphenol A.

Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro

Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes.

Development and validation of a hydrophilic interaction chromatography–tandem mass spectrometry for quantification of nicotine and its metabolites in human maternal and cord sera

A selective and sensitive HILIC-MS/MS method for the simultaneous quantification of nicotine and its metabolites in human maternal and cord sera was developed and validated. After solid-phase extraction, LC separation was achieved on a hydrophilic interaction chromatography. The validated method is capable of selective identification as well as accurate and sensitive quantification. Analyte recovery ranged from 86.2 to 107.7% and intra- and inter-day assay precision were less than 15% relative standard deviation. This sensitive HILIC-MS/MS method can be used to determine nicotine and its metabolic profile in smokers. This validated method is useful for the determination of nicotine and its metabolites in human serum in future studies of the effects of nicotine exposure on neonatal outcome.