Maternal and Neonatal Factors' Effects on Wharton's Jelly Mesenchymal Stem Cell Yield

Maternal and Neonatal Factors' Effects on Wharton's Jelly Mesenchymal Stem Cell Yield | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Maternal and Neonatal Factors' Effects on Wharton's Jelly Mesenchymal Stem Cell Yield Ranim Mahmoud, Mohamed Bassiouny, Ahmed Badawy, Ahmad Darwish, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4437032/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Because Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are easily accessible, easy to isolate, and ethically acceptable, they represent a talented source of MSCs for use in regenerative medicine. Considering decisions on WJ-MSCs collection requires extensive knowledge of the factors that impact their yield. This study's objective was to evaluate the influence of parameters related to mothers and newborns on the WJ-MSCs yield. The WJ-MSCs were isolated and expanded after being taken from 79 umbilical cord samples. Population doubling time and cell proliferation were assessed. By flow cytometry analysis, WJ-MSCs were identified by positivity of CD105, CD90, and CD73 and negativity of CD45 and CD34. There was a statistically significant negative correlation between umbilical cord width and P1 doubling time. Maternal age and WJ-MSC yield were shown to be negatively correlated. Birth weight and gestational age showed a significant positive correlation between WJ-MSCs yield and neonatal variables. No significant correlations were detected between the WJ-MSCs and the mother parity, nor the neonatal sex, fetal presentation, or head circumference. The WJ-MSCs yield increases with younger maternal age, increased gestational age, and increased neonatal birth weight. Hence, consideration should be given to these factors when selecting the ideal donors. Biological sciences/Stem cells Health sciences/Medical research Wharton's jelly Stem cells Yield Maternal- Neonatal Flow cytometry Figures Figure 1 Figure 2 Introduction The stem cells are a special type of cells that have a potentiality for regeneration and thus can maintain and repair cellular damage. So, it have a promising therapeutic prospective in cell transplantation. Mesenchymal stem cells (MSCs), hematopoietic stem cells, and other progenitors are among the cells from different origins that have been isolated Anthony and Shiels [ 1 ] [ 2 ]. Mesenchymal stem cells (MSCs) isolated from bone marrow have been the focus of most studies; however, MSCs may also be obtained from amniotic fluid and umbilical cord (UC) blood [ 3 ] and chorionic villi [ 4 ]. The mesenchymal stem cells have been also derived from the Wharton's jelly, the connective tissue that envelops the two arteries and vein in the UC [ 5 ]. WJ-MSCs have grown increasingly common in recent years due to their inherent advantages than MSCs obtained from other sources (Baksh et al., 2007). These advantages include increased ex-vivo growth capabilities, faster proliferation, a lower incidence of graft versus host medical problems, and a lower risk of teratomas [ 6 ]. Additionally, there are no ethical issues involved in obtaining them, in contrast to bone marrow and embryonic stem cells, which are often thrown as human waste [ 7 ]. Wharton's jelly mesenchymal stem cells (WJ-MSCs) possess paracrine activity, which is the ability to alter their environment by releasing bioactive chemicals and compounds known as secretome. This activity is the primary reason for the therapeutic potential of WJ-MSCs. The secretome has anti-inflammatory and anti-fibrotic properties, but it also carries out several biological activities like immuno-modulation, tissue replenishment, and cellular homeostasis [ 8 ]. Previous researches recommended its use for cancer cells growth inhibition [ 9 ], ameliorating diabetic nephropathy and hepatopathy complications as a novel protective approach [ 10 ], the reduction of the severity of the inflammation and immune response against Covid-19 because of its anti-inflammatory and immunomodulatory characteristics [ 11 ], and reduce fibrosis caused by bleomycin-induced lung injury [ 12 ]. There are many approaches to identifying mesenchymal stem cells as their capacity to adhere to flexible surfaces and their spindle-like appearance in culture and they can differentiated into adipocytes, chondrocytes and osteocytes. Furthermore, WJ-MSCs should express CD105, CD73 and CD90 and not display CD34, CD45, CD19 or CD11b, CD79 alpha or CD14, or HLA-DR surface molecules, according to the International Society for Cellular Therapy (ISCT) [ 13 ]. Since isolating cells mostly implies an invasive and technically challenging procedure that affects the quality and biological activity of the derived cells and restricts their ability to proliferate and differentiate during in vitro culture, the quantity of retrieved stem cells is inadequate [ 14 ]. It is essential to gain a good yield from the WJ-MSCs. Many factors have been suggested to impact the quantity of WJ-MSCs isolated from Wharton’s jelly, and which may be accountable for the variations in the reported results. These variables include the mother's age, the gestational age, mother’s parity and gravidity, the newborn's sex, and the birth weight, all of which might affect the amount of CD34 + cells in the blood [ 15 ]. Considering the maternal and neonatal factors in the selection of promising donors for isolation of a good yield from WJ-MSCs, our study aimed to determine whether maternal parameters-such as the mother's age, gravidity, parity, and other health-related conditions—and neonatal parameters—such as birth weight, sex, head circumference, and UC width—may affect the yield of WJ-MSCs. Patients and Methods The Institutional Review Board Mansoura Faculty of Medicine, Mansoura University, Egypt approved the current study, and informed consents were obtained from all participants before delivery. A total of 100 pregnant women were included in the study. Women with temperature higher than 38 o C, having premature rupture of membranes, with multiple gestation, positive for blood transmitted infections or with proved fetal malformations were excluded from the study. The patient records were reviewed for information on pregnancy history, number of prior pregnancies, parities, the age of gestation, newborn sex, newborn weight, and head circumference, the width of UC, gestational diabetes, pre-eclampsia, and any reports of maternal co-morbidity. Patients' records and gathered data for the study were kept confidential and experiments were performed in accordance with relevant guidelines and regulations. Samples collection and cord processing: Following cesarean birth, a total of 100 fresh human UC samples were collected. A collection cup holding in Dullbecco's Modified Eagle Media (DMEM) with 4,500 mg/mL glucose and antibiotic solution (.2% streptomycin,, .12% penicillin and .1% gentamicin) (Lonza, Belgium) was used to hold the roughly 5 gm of UC that were aseptically collected. The samples were subsequently kept at 4°C. After that, the samples were sent to the Mansoura University Stem Cell Research Center lab to be processed. In order to eliminate blood clots, samples were processed in a biosafety cabinet and washed using ice-cold phosphate buffered saline repeatedly. The explant method was utilized to isolate mesenchymal stem cells from the UC Wharton's jelly [ 16 ]. Six to nine pieces of the explant outgrowth are cut off and put on the culture dishes, where they remain until the jelly hardens. Next, let's include a culture media (DMEM). The culture dishes are kept for 3 to 4 days at 37°C in a humidified atmosphere with 5% CO2. Following the attachment of Wharton jelly, the dish media is replaced every 2 to 3 days, and after about 7 to 10 days, the cells displayed the WJ-MSCs phenotype will be recovered. The cells were separated by the use of 1–2 mL of commercial trypsin solution in a 25-cm 2 culture flask before being incubated for 3 min. at 37°C. Using a tissue culture centrifuge, the mixture was centrifuged at 1,500×g for three minutes in order to neutralize the trypsin. One to two milliliters of culture medium were added. To carry out amplification and characterization, the pelleted cells—which are regarded as passage 0 (P0)—were re-suspended in a culture medium, counted, and sub-cultured at a 1x10 4 /cm 2 seeding density. Cell viability determination and Population Doubling Time determination: The cell viability was determined using the Trypan blue test as described by [ 17 ]. After the isolated cells were stained with Trypan blue, the number of viable (white) and non-viable (blue) cells was determined using a hemocytometer composed of nine 1×1 mm (1mm 2 ) squares and a microscope (AX-71, Olympus Corporation, Shinjuku-ku, Japan). Every experiment was carried out three times in duplicate. Trypsin was used to harvest the cells at 80–90% confluence after each passage. The cells were then plated in a T25 cell culture flask, counted, and re-plated till the third passage. The population doubling time (PDT) was calculated according to [ 18 ]. The following formula was used for calculating PDT, which was expressed in hours: PDT is equal to (lgNt – lgN0)/lg2, where t is the culture period, Nt is the harvested cell count after the cell passage, and N0 is the number of cells seeded at the start of the passage. Flow cytometry: Using the International Society for Hematotherapy and Graft Engineering (ISHAGE) procedure [ 19 ], flow cytometric analysis was used to determine if the isolated cells were multipotent WJ-MSCs and to evaluate the WJ-MSCs yield. The BD Accuri TM C6 Cytometer (Becton, Dickinson and Company) was used. The program System (Becton, Dickinson and Company) was used for analyzing the data [ 20 ]. According to [ 21 ], the WJ-MSCs were characterized by positivity of CD73, CD90, CD105 and negativity of CD34 and CD45 on flow cytometry analysis as shown in figure (1). In brief, after being re-suspended in 1 ml of PBS including 1% FBS, the cultured WJ-MSCs were labeled with 5µl of fluorescein isothiocyanate (FITC), APC anti-human, and phycoerythrin (PE)-conjugated antibodies as positive markers for CD105, CD90, and CD73 respectively. Antibodies conjugated with phycoerythrin cyanine 5 (PE-Cy5) and PE-Cy7 were utilized as negative markers for CD45 and CD34, respectively. As controls, FITC, PE, and PC5 matched isotype antibodies were used. All antibodies were from BD Biosciences except CD90,and CD73 were from Miltenyil Biotec. At 4°C and in the dark for 45 minutes, antibodies were added at 1:1000 dilutions then the yield was assessed using flow cytometry. Statistical analysis: The collected data was revised, coded, tabulated and introduced to Statistical package for Social Science (IBM SPSS Statistics, Version 25.0. Armonk, NY). Mann Whitney-U or student t-test was applied for comparing numerical variables between groups. Chi-square test was utilized to compare categorical variables. Correlation analysis was used for studying the strength of correlation between two numerical variables. The correlation between WJ-MSCs yield at the end of P3 and P1 doubling time and the potential numerical maternal and newborn predictors was tested by Spearman correlation coefficient. A correlation coefficient of less than .3, between .3-.7 and of more than .7 indicated weak, moderate and strong linear correlations, respectively. Results Demographics: At the Mansoura University Hospital delivery unit, UC samples were obtained from 100 singleton newborns who were delivered via Caesarean section. Twenty-one specimens were excluded because, in 14 samples, the material was contaminated, and in 7 samples, there was no apparent cellular growth after 20 days. The final analysis had 79 specimens in total. The maternal data were as follows: the mean parity was 1.2 ± 1.1, the mean gravidity was 2.4 ± 1.3, and the mean age was 29.3 ± 5.4 years, with a range of 18 to 42 years. Four (5.1%) of the mothers in the study have gestational diabetes; two were managed with diet, and the other four required insulin therapy. There were six mothers (7.6%) with pre-eclampsia. Of the seventy-nine babies who were part of the study, fifty-one (64.6%) were female and twenty-eight (35.4%) were male. Of the infants born between 26 and 41 weeks, 56 (70.9%) were full term and 23 (29.5%) were preterm. The mean gestational age was (36.3 ± 3.3). The median birth weight was 2.8 (1.3–4) kg, with a mean of 2.73 ± .6 kg. The median birth length was 45 (39–52) cm, with a mean of 45.5 ± 2.99 cm. Within the 30–35 range, the average newborn head circumference was 33.1 ± 4.8. Ten (12.7%) newborns had amniotic fluid stained with meconium, while sixty (75.9%) babies were cephalic presentation. Effects of neonatal and maternal variables on the passage (P1) doubling time: For WJ-MSCs, the average P1 doubling time was 68 (55–90) hours. When the association between P1 doubling time and maternal variables were analyzed, Fig. 2 A, 2 B, and 2 C showed that P1 doubling time had a small positive correlation with gravidity (r = .624, P < .001), parity (r = .551, P < .001), and a strong positive correlation with maternal age (r = .827, P < .001). P1 doubling time was not significantly associated with any of the neonatal variables: birth head circumference ( P = .543), birth length ( P = .236), birth weight ( P = .735), or gestational age ( P = .186). There was no statistically significant difference in the median doubling time between preterm newborns (69 hours) and full term babies (67.5 hours) ( P = .612). In addition, there was no statistically significant difference in the hours of male (66) and female (68) babies ( P = .612). The presence of amniotic fluid stained with meconium ( P = .238) or fetal mal-presentation ( P = .502) had no discernible effect on the P1 doubling time. Double time and UC width were shown to have a statistically significant negative connection (r=-.5, P = .001) (Fig. 2 D). Maternal and neonatal variables' effects on the yield of WJ-MSCs: By the end of P3, the mean WJ-MSCs yield was (2.34 ± .74×106) cell/ml [2.6 (.4–3.5) ×106 cell/ml, according to the median (range). For the median (range), the viability percentage was 83.1 ± 7.6% [84.5 (65–97)%]. UC samples were used to determine the WJ-MSCs count of full-term neonates (2.37 ± .74 x 106 cell/ml; median = 2.6 (.4–3.5)×106 cell/ml) and preterm newborns (2.27 ± .76 x 106 cell/ml; median = 2.6 (.7–3.3)×106 cell/ml). No statistically significant difference was seen in the median WJ-MSCs yield between preterm and full-term newborns (Mann-Whitney Up = .742). Maternal age and WJ-MSCs yield had a negative correlation (r=-.246, P = .029) according to an analysis of maternal variables (Table 1). There was no statistically significant difference in the median WJ-MSCs yield between UC specimens obtained from women with and without gestational diabetes. In UC specimens collected from mothers with or without pre-eclampsia, WJ-MSCs yield was also similar. It is noteworthy, therefore, that there were very few pre-eclamptic or gestational diabetic individuals in the study. WJ-MSCs yield did not significantly correlate with maternal hemoglobin level prior to birth, parity, or gravidity. Analyses of the association between WJ-MSCs yield and newborn characteristics (Table 1) revealed a positive correlation between the WJ-MSCs count and birth weight and gestational age. However, it was shown that the yield of WJ-MSCs and UC width had a weakly positive correlation. A multivariate linear regression analysis revealed that the sole independent predictor of WJ-MSCs production was maternal age (Table 2). The yield of WJ-MSCs was not independently predicted by gestational age, birth weight, or cord breadth (P = 0.330). Discussion A promising source of MSCs for therapeutic uses is Wharton's Jelly. WJ-MSCs are easily accessible with many advantages over the BM-MSCs. Several maternal and neonatal parameters have been studied in relation to the characterization and quality of hematopoietic UC blood stem cells in the past. Although research on how maternal and neonatal variables affect the yield of WJ-MSCs is still under research investigation. For the purpose of aiding in finding and selecting the best UC donors, this study studied the effects of many maternal and fetal factors on the yield of WJ-MSCs. According to our findings, the mother's age has a substantial impact on the yield of WJ-MSCs. This is in agreement with the findings of [ 22 ], who found that whereas UC-MSCs from elder donors show diminished differentiation potential, UC from younger donors is a reasonably abundant source of MSCs. Furthermore, compared to younger age groups, there was a significant negative correlation in the expression levels of both CD29 and CD105, according to [ 23 ]. It was reported that as a woman ages, her mesenchymal stem cells expressing the BIRC2 and BIRC3 genes decrease as stem cells obtained from younger women may exhibit more apoptotic resistance and a more stem cell-like quality, which may enhance their therapeutic potential and clinical usability [ 16 ]. A known pluripotent transcription factor, SOX2 is implicated in reprogramming, self-renewal, and maintaining the homeostasis of stem cells. The SOX2 gene is statistically considerably more expressed in WJSC in women under the age of 34 than it is in women over 34, according to [ 24 ]. Furthermore, a statistically significant moderate negative correlation is demonstrated between the SOX2 gene expression and the maternal age. This result was clarified by [ 25 ] and [ 26 ], who found that aging in mothers is related to telomere shortening and increased susceptibility to apoptosis, and that stem cells' telomere length could not be maintained by the level of telomerase activity. These results largely stated the decline in CD105, CD73, and CD90 expression levels that they reported in their study's UC-MSCs as they were older. Furthermore, it has been previously reported how aging affects adult stem cells. [ 27 ] found a decrease in adult MSCs number derived from the bone marrow with aging. According to [ 28 ], there is a decrease in the quantity, functionality, and multi-lineage differentiation characteristics of mature MSCs as they age. Additionally, the authors hypothesized that the lower differentiation capacity of the older donor's umbilical cord cells could be related to the declining functional state of the mother's older organs, which support and foster the development of umbilical cord MSC [ 22 ]. Conversely to this finding, maternal age had no effect on the quality of WJ-MSCs, according to [ 29 ]. According to [ 30 ], vaginal and cesarean births had MSC cell yields that were comparable. According to [ 29 ], the method of delivery had no impact on the characteristics or survivability of MSCs. To reduce the expectation of contamination, we only used specimens from Cesarean births in our investigation. We observed that the WJ-MSCs count and doubling time were not more impacted by birth order. A reduction in the number of WJ-MSCs was correlated with increasing parity, nevertheless the relationship was not statistically significant. Birth order and viability showed a moderately positive association (r = .119, p = .044), whereas parity and the amount of UC blood showed no correlation (P = .057) [ 31 ]. According to [ 32 ], WJ-MSCs obtained from younger women who gave vaginal delivery have increased expression of the BIRC2, BIRC3, and BIRC5 genes. Pre-eclampsia had no obvious effect on the population doubling time or WJ-MSC yield in this study. In contrast, pre-eclampsia has been shown by [ 33 ] to result in a considerable increase in cell count and reduction in the fraction of immature cells and increasing the mature cells. According to [ 34 ] compared to normal samples, the proliferation rate of early-passage gestational diabetes mellitus (DM) samples was significantly lower. Furthermore, with a mean cell density of 1.9 x 10 4 cells per plate in gestational DM samples as opposed to 4.1×10 4 cells per plate in normal samples, the population doubling rate was considerably lower in these samples after 12 days of incubation. Gestational DM-UC-MSCs did not grow above passages 6 or 7. All UC-MSCs had comparable amounts of the stem cell markers CD90, CD73, and CD105 during early stages. These results are consistent with a study conducted by [ 35 ], which found that MSCs derived from Wharton Jelly of the DM group grew and proliferated at a much slower rate in all passages than MSCs isolated from a control group. The quantity of non-viable cells in the gestational DM samples significantly increased than viable cells. The WJ-MSCs count was shown to be statistically significantly impacted by gestational age in our investigation, although there was not a significant association established between gestational age and P1 doubling time. Nevertheless, there was no statistically significant difference in the median MSC output between full-term and preterm newborn when comparing them to full-term babies. In a study by [ 36 ] divided 45 babies into three groups based on their gestational ages: group A (28–31 weeks), group B (32–35 weeks), and group (C) ≥ 36 weeks. The authors observed a significant inverse relationship between gestational age and MSC production, even after controlling for birth weight, the percentage of small for gestational age neonates, maternal co-morbidities, and prenatal steroid usage. Similar findings were made by [ 37 ], who found that despite both samples maintained a high rate of proliferation, preterm cord samples developed more MSC in all passages than term cords. On the other hand, according to [ 38 ], the quantity of umbilical cord vein UC-MSCs from both full term and preterm patients is comparable, and both groups exhibit a high rate of proliferation. [ 29 ], on the other hand, found no correlation between gestational age and MSC yield. Age at gestation may possibly have an impact on the UC-MSCs' potential to modulate immunity. According to [ 38 ], preterm UC-MSCs do not have the same potent inhibitory effects on immune cell proliferation as full-term UC-MSCs. A lymphocyte co-culture experiment revealed that both full term and preterm UC-MSCs reduced peripheral blood mononuclear cell proliferation in the mixed lymphocyte response. Compared to preterm UC-MSCs, the term UC-MSCs had a stronger proliferation inhibition. The scientists explained the inhibitory difference by the preterm UC-MSCs' immature immune systems. The source, donor age, and passage number of cells can also have an impact on the immuno-modulatory qualities of MSCs [ 39 ] and [ 40 ]. According to [ 41 ], placental weight, birth weight, and full term birth were the primary factors influencing cell proliferation. Despite the strong correlation between these parameters and gestational age, multivariate regression analysis revealed that gestational age was the only independent predictor of population doubling time (P = .0094). Our study found that the baby's birth weight had a substantial impact on the number of WJ-MSCs; a greater birth weight leads to increased the yield of MSCs. According to [ 30 ], the yield of MSCs was not significantly affected by the mother's age or gestational age, but a greater birth weight was associated with a more yield, which is consistent with our findings. On the other hand,[ 24 ] found that lower birth weight correlates to higher SOX2 gene expression in WJ-SCs. SOX2 is a known pluripotent transcription factor that contributes to cell reprogramming and self-renewal. According to [ 29 ], the MSCs output was not significantly impacted by birth weight. The author explained that his study focused on the quality of the stem cells not the number so the study finding differs from other studies that correlate the number of isolated stem cells to the birth weight [ 31 ] [ 42 ]. According to our findings, there was no statistically significant variation in the WJ-MSCs yields with respect to fetal gender, presentation, length at birth, or head circumference. According to [ 36 ], the mean MSCs number of female neonates was greater than that of male infants. When comparing MSCs separated from WJ from female individuals to MSCs obtained from WJ from male subjects, [ 43 ] observed that the expression of OCT4 and DNMT1 genes was considerably greater in WJ-MSCs derived from male participants. They came to the conclusion that gender significantly affects stemness genes. However, [ 29 ] found that gender did not affect the quality of WJ-MSCs. We discovered a statistically significant negative association between doubling time and UC width and a slight positive correlation between MSCs yield and UC width. We discovered that the amount of meconium in the amniotic fluid had no effect on the production of MSCs or the population doubling time. According to [ 44 ], there was little change in the expression of the mesenchymal markers CD90 and CD105 following exposure to meconium. Amniotic fluid MSCs may change in phenotype after being exposed to meconium, although they are still capable of differentiating. To sum up, Wharton's jelly MSCs are a perfect and exciting source of MSCs. Numerous factors related to mothers and newborns may impact the MSC output and should be investigated for settling the optimal selection criteria for the ideal donors. Our research showed that a higher yield of MSCs produced from Wharton's jelly correlates to a younger mother's age, increased birth weight, and longer gestational age. Declarations Competing Interests: The authors declare no competing interests. Author Contribution B.M., B.A. and Y.S. designed and supervised the work; M.R, D.A. and Y.S. performed the experiment. B.M., B.A. and M.R. performed the analysis; D.A., E.N, M.R. and Y.S. drafted the manuscript and designed the figures. B.M., B.A. and Y.S. aided in interpreting the results and worked on the manuscript. All authors discussed the results and commented on the manuscript. Data Availability "The data that support the findings of this study are available on request from the corresponding author, E.N." References Anthony, D.F. and P.G. Shiels, Exploiting paracrine mechanisms of tissue regeneration to repair damaged organs . Transplantation research, 2013. 2: p. 1–8. Zimran, E., et al., Expansion and preservation of the functional activity of adult hematopoietic stem cells cultured ex vivo with a histone deacetylase inhibitor . 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Tables Table (1): A summary of the correlation between neonatal and maternal variables and the yield of WJ-MSCs Maternal variables Correlation coefficient P value Mother's age (years) -.246* .029* Mother's hemoglobin level (gm/dl) .037 .743 Parity .069 .545 Gravidity .003 .982 Toxemia .732** Gestational diabetes .762** Neonatal variables Gestational age in weeks .329 .004* Sex .681** Fetal presentation .373** Newborn birth weight (kg) .272 .015* The width of Umbilical Cord (cm) .285 .011* Birth length (cm) .057 .620 The head circumference (cm) .130 .253 Meconium stained amniotic fluid .308** P values < .05*, **Mann-Whitney U test. Table (2): The WJ-MSCs yield model using linear regression analysis. Variables Standardized ẞ P -value The age of gestation -.151 .563 Maternal age -.249 .027 Neonate birth weight .302 .051 The width of Umbilical Cord .210 .330 (Adjusted P =.003, R 2 =.146). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 17 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Jun, 2024 Reviews received at journal 07 Jun, 2024 Reviews received at journal 27 May, 2024 Reviewers agreed at journal 23 May, 2024 Reviewers agreed at journal 23 May, 2024 Reviewers invited by journal 23 May, 2024 Editor assigned by journal 23 May, 2024 Editor invited by journal 22 May, 2024 Submission checks completed at journal 21 May, 2024 First submitted to journal 17 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4437032","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":307107205,"identity":"f0aa9a26-4299-4121-bd62-41abae4228fb","order_by":0,"name":"Ranim Mahmoud","email":"","orcid":"","institution":"Pediatric Department, Mansoura University Children’s Hospital, Faculty of Medicine, Mansoura University, Mansoura","correspondingAuthor":false,"prefix":"","firstName":"Ranim","middleName":"","lastName":"Mahmoud","suffix":""},{"id":307107207,"identity":"7c8b81d5-8437-45e6-a043-1cd0f8d60887","order_by":1,"name":"Mohamed Bassiouny","email":"","orcid":"","institution":"Pediatric Department, Mansoura University Children’s Hospital, Faculty of Medicine, Mansoura University, Mansoura","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Bassiouny","suffix":""},{"id":307107209,"identity":"9b065f0d-cf45-4602-8150-4cfbc87f8d73","order_by":2,"name":"Ahmed Badawy","email":"","orcid":"","institution":"Obstetric and Gynecology Department, Faculty of Medicine, Mansoura University, Mansoura","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Badawy","suffix":""},{"id":307107211,"identity":"3e186030-47b2-40de-92a8-17da736f670c","order_by":3,"name":"Ahmad Darwish","email":"","orcid":"","institution":"Pediatric Department, Mansoura University Children’s Hospital, Faculty of Medicine, Mansoura University, Mansoura","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Darwish","suffix":""},{"id":307107212,"identity":"69304ad9-66ad-4639-bf8b-932be90e34cc","order_by":4,"name":"Sohier Yahia","email":"","orcid":"","institution":"Pediatric Department, Mansoura University Children’s Hospital, Faculty of Medicine, Mansoura University, Mansoura","correspondingAuthor":false,"prefix":"","firstName":"Sohier","middleName":"","lastName":"Yahia","suffix":""},{"id":307107214,"identity":"f87a9d37-f194-46ae-b029-2273acf420ca","order_by":5,"name":"Nora El-Tantawy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYLCCBwwHGBgkmA9AuQlEaEkAa2FLgHOJ1cJjQJwWc/behx8Sau7I8Uv3fN3wc0cdAz97jgHDh1+4tVj2HDeWSDj2zFhyztltN3vPHGaQ7HljwDizD7cWgxtpDBIJbIcTN9zI3XaDt+0AUCTHgJm3B68W5h8J/w7X77+R8+zm37Y6BnuQlr/4tbBJJLYdTjCQyGG7zdvGzABkGDAz/MCj5cwxNovEvmeGM+4cM7st23aYR+LMs4KDvQ14tBxvY77x4dsdef7Zzc9uvm2rk+NvT9744Mcf3FowAA+IOMDYRoIWKCDFllEwCkbBKBjuAADS4lwP9nqZfQAAAABJRU5ErkJggg==","orcid":"","institution":"Medical Parasitology Department, Faculty of Medicine, Mansoura University, Mansoura","correspondingAuthor":true,"prefix":"","firstName":"Nora","middleName":"","lastName":"El-Tantawy","suffix":""}],"badges":[],"createdAt":"2024-05-17 13:29:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4437032/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4437032/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-72386-z","type":"published","date":"2024-10-17T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57865581,"identity":"e5318285-8304-41b3-9661-cdc885ea0e4c","added_by":"auto","created_at":"2024-06-06 15:43:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67184,"visible":true,"origin":"","legend":"\u003cp\u003eFlow Cytometry Analysis\u003c/p\u003e\n\u003cp\u003e(A) shows the negative expression of CD34 (hematopoietic stem cell marker) on WJ-MSCs lineage, (B) shows two parameters plots for WJ-MSCs labeled with CD90 and CD105, where Q1 denotes cells positive for CD90, Q2 double-positive cells, Q3 positive cells for CD105, and Q4 negative cells for both markers.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4437032/v1/58333f9e9e7aa4df0188cedb.jpg"},{"id":57865582,"identity":"32c5a962-e718-40e0-88d5-cb6a0f0609eb","added_by":"auto","created_at":"2024-06-06 15:43:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160539,"visible":true,"origin":"","legend":"\u003cp\u003eP1 doubling time and correlation between variables\u003c/p\u003e\n\u003cp\u003e(A) shows a scatter plot demonstrating the correlation between WJ-MSCs P1 maternal age and doubling time (P\u0026lt;.001, r =.827,), (B) a box plot demonstrating the correlation between P1 doubling time and gravidity (P\u0026lt;.001, r=.624), (C) a box plot demonstrating the relationship between P1 doubling time and parity (P\u0026lt;.001, r = .551), and (D) shows a scatter plot showing the correlation between umbilical cord width and P1 doubling time.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4437032/v1/e903b6c297e7b05404457db9.jpg"},{"id":67148936,"identity":"5277dd4c-a986-4990-b0ef-90397749711c","added_by":"auto","created_at":"2024-10-21 16:10:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":595615,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4437032/v1/8137dcbc-e22a-4091-89ac-708f991b14fa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Maternal and Neonatal Factors' Effects on Wharton's Jelly Mesenchymal Stem Cell Yield","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe stem cells are a special type of cells that have a potentiality for regeneration and thus can maintain and repair cellular damage. So, it have a promising therapeutic prospective in cell transplantation. Mesenchymal stem cells (MSCs), hematopoietic stem cells, and other progenitors are among the cells from different origins that have been isolated Anthony and Shiels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mesenchymal stem cells (MSCs) isolated from bone marrow have been the focus of most studies; however, MSCs may also be obtained from amniotic fluid and umbilical cord (UC) blood [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and chorionic villi [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The mesenchymal stem cells have been also derived from the Wharton's jelly, the connective tissue that envelops the two arteries and vein in the UC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWJ-MSCs have grown increasingly common in recent years due to their inherent advantages than MSCs obtained from other sources (Baksh et al., 2007). These advantages include increased ex-vivo growth capabilities, faster proliferation, a lower incidence of graft versus host medical problems, and a lower risk of teratomas [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, there are no ethical issues involved in obtaining them, in contrast to bone marrow and embryonic stem cells, which are often thrown as human waste [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Wharton's jelly mesenchymal stem cells (WJ-MSCs) possess paracrine activity, which is the ability to alter their environment by releasing bioactive chemicals and compounds known as secretome. This activity is the primary reason for the therapeutic potential of WJ-MSCs. The secretome has anti-inflammatory and anti-fibrotic properties, but it also carries out several biological activities like immuno-modulation, tissue replenishment, and cellular homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous researches recommended its use for cancer cells growth inhibition [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], ameliorating diabetic nephropathy and hepatopathy complications as a novel protective approach [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], the reduction of the severity of the inflammation and immune response against Covid-19 because of its anti-inflammatory and immunomodulatory characteristics [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and reduce fibrosis caused by bleomycin-induced lung injury [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are many approaches to identifying mesenchymal stem cells as their capacity to adhere to flexible surfaces and their spindle-like appearance in culture and they can differentiated into adipocytes, chondrocytes and osteocytes. Furthermore, WJ-MSCs should express CD105, CD73 and CD90 and not display CD34, CD45, CD19 or CD11b, CD79 alpha or CD14, or HLA-DR surface molecules, according to the International Society for Cellular Therapy (ISCT) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Since isolating cells mostly implies an invasive and technically challenging procedure that affects the quality and biological activity of the derived cells and restricts their ability to proliferate and differentiate during in vitro culture, the quantity of retrieved stem cells is inadequate [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It is essential to gain a good yield from the WJ-MSCs. Many factors have been suggested to impact the quantity of WJ-MSCs isolated from Wharton\u0026rsquo;s jelly, and which may be accountable for the variations in the reported results. These variables include the mother's age, the gestational age, mother\u0026rsquo;s parity and gravidity, the newborn's sex, and the birth weight, all of which might affect the amount of CD34\u0026thinsp;+\u0026thinsp;cells in the blood [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering the maternal and neonatal factors in the selection of promising donors for isolation of a good yield from WJ-MSCs, our study aimed to determine whether maternal parameters-such as the mother's age, gravidity, parity, and other health-related conditions\u0026mdash;and neonatal parameters\u0026mdash;such as birth weight, sex, head circumference, and UC width\u0026mdash;may affect the yield of WJ-MSCs.\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cp\u003eThe Institutional Review Board Mansoura Faculty of Medicine, Mansoura University, Egypt approved the current study, and informed consents were obtained from all participants before delivery. A total of 100 pregnant women were included in the study. Women with temperature higher than 38\u003csup\u003eo\u003c/sup\u003eC, having premature rupture of membranes, with multiple gestation, positive for blood transmitted infections or with proved fetal malformations were excluded from the study. The patient records were reviewed for information on pregnancy history, number of prior pregnancies, parities, the age of gestation, newborn sex, newborn weight, and head circumference, the width of UC, gestational diabetes, pre-eclampsia, and any reports of maternal co-morbidity. Patients' records and gathered data for the study were kept confidential and experiments were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003cp\u003eSamples collection and cord processing:\u003c/p\u003e \u003cp\u003eFollowing cesarean birth, a total of 100 fresh human UC samples were collected. A collection cup holding in Dullbecco's Modified Eagle Media (DMEM) with 4,500 mg/mL glucose and antibiotic solution (.2% streptomycin,, .12% penicillin and .1% gentamicin) (Lonza, Belgium) was used to hold the roughly 5 gm of UC that were aseptically collected. The samples were subsequently kept at 4\u0026deg;C. After that, the samples were sent to the Mansoura University Stem Cell Research Center lab to be processed. In order to eliminate blood clots, samples were processed in a biosafety cabinet and washed using ice-cold phosphate buffered saline repeatedly.\u003c/p\u003e \u003cp\u003eThe explant method was utilized to isolate mesenchymal stem cells from the UC Wharton's jelly [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Six to nine pieces of the explant outgrowth are cut off and put on the culture dishes, where they remain until the jelly hardens. Next, let's include a culture media (DMEM). The culture dishes are kept for 3 to 4 days at 37\u0026deg;C in a humidified atmosphere with 5% CO2. Following the attachment of Wharton jelly, the dish media is replaced every 2 to 3 days, and after about 7 to 10 days, the cells displayed the WJ-MSCs phenotype will be recovered. The cells were separated by the use of 1\u0026ndash;2 mL of commercial trypsin solution in a 25-cm\u003csup\u003e2\u003c/sup\u003e culture flask before being incubated for 3 min. at 37\u0026deg;C. Using a tissue culture centrifuge, the mixture was centrifuged at 1,500\u0026times;g for three minutes in order to neutralize the trypsin. One to two milliliters of culture medium were added. To carry out amplification and characterization, the pelleted cells\u0026mdash;which are regarded as passage 0 (P0)\u0026mdash;were re-suspended in a culture medium, counted, and sub-cultured at a 1x10\u003csup\u003e4\u003c/sup\u003e/cm\u003csup\u003e2\u003c/sup\u003e seeding density.\u003c/p\u003e \u003cp\u003eCell viability determination and Population Doubling Time determination:\u003c/p\u003e \u003cp\u003eThe cell viability was determined using the Trypan blue test as described by [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. After the isolated cells were stained with Trypan blue, the number of viable (white) and non-viable (blue) cells was determined using a hemocytometer composed of nine 1\u0026times;1 mm (1mm\u003csup\u003e2\u003c/sup\u003e) squares and a microscope (AX-71, Olympus Corporation, Shinjuku-ku, Japan). Every experiment was carried out three times in duplicate.\u003c/p\u003e \u003cp\u003eTrypsin was used to harvest the cells at 80\u0026ndash;90% confluence after each passage. The cells were then plated in a T25 cell culture flask, counted, and re-plated till the third passage. The population doubling time (PDT) was calculated according to [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The following formula was used for calculating PDT, which was expressed in hours: PDT is equal to (lgNt \u0026ndash; lgN0)/lg2, where t is the culture period, Nt is the harvested cell count after the cell passage, and N0 is the number of cells seeded at the start of the passage.\u003c/p\u003e \u003cp\u003eFlow cytometry:\u003c/p\u003e \u003cp\u003eUsing the International Society for Hematotherapy and Graft Engineering (ISHAGE) procedure [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], flow cytometric analysis was used to determine if the isolated cells were multipotent WJ-MSCs and to evaluate the WJ-MSCs yield. The BD Accuri TM C6 Cytometer (Becton, Dickinson and Company) was used. The program System (Becton, Dickinson and Company) was used for analyzing the data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the WJ-MSCs were characterized by positivity of CD73, CD90, CD105 and negativity of CD34 and CD45 on flow cytometry analysis as shown in figure (1).\u003c/p\u003e \u003cp\u003eIn brief, after being re-suspended in 1 ml of PBS including 1% FBS, the cultured WJ-MSCs were labeled with 5\u0026micro;l of fluorescein isothiocyanate (FITC), APC anti-human, and phycoerythrin (PE)-conjugated antibodies as positive markers for CD105, CD90, and CD73 respectively. Antibodies conjugated with phycoerythrin cyanine 5 (PE-Cy5) and PE-Cy7 were utilized as negative markers for CD45 and CD34, respectively. As controls, FITC, PE, and PC5 matched isotype antibodies were used. All antibodies were from BD Biosciences except CD90,and CD73 were from Miltenyil Biotec. At 4\u0026deg;C and in the dark for 45 minutes, antibodies were added at 1:1000 dilutions then the yield was assessed using flow cytometry.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eThe collected data was revised, coded, tabulated and introduced to Statistical package for Social Science (IBM SPSS Statistics, Version 25.0. Armonk, NY). Mann Whitney-U or student t-test was applied for comparing numerical variables between groups. Chi-square test was utilized to compare categorical variables. Correlation analysis was used for studying the strength of correlation between two numerical variables. The correlation between WJ-MSCs yield at the end of P3 and P1 doubling time and the potential numerical maternal and newborn predictors was tested by Spearman correlation coefficient. A correlation coefficient of less than .3, between .3-.7 and of more than .7 indicated weak, moderate and strong linear correlations, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eDemographics:\u003c/p\u003e \u003cp\u003eAt the Mansoura University Hospital delivery unit, UC samples were obtained from 100 singleton newborns who were delivered via Caesarean section. Twenty-one specimens were excluded because, in 14 samples, the material was contaminated, and in 7 samples, there was no apparent cellular growth after 20 days. The final analysis had 79 specimens in total. The maternal data were as follows: the mean parity was 1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1, the mean gravidity was 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3, and the mean age was 29.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 years, with a range of 18 to 42 years. Four (5.1%) of the mothers in the study have gestational diabetes; two were managed with diet, and the other four required insulin therapy. There were six mothers (7.6%) with pre-eclampsia. Of the seventy-nine babies who were part of the study, fifty-one (64.6%) were female and twenty-eight (35.4%) were male. Of the infants born between 26 and 41 weeks, 56 (70.9%) were full term and 23 (29.5%) were preterm. The mean gestational age was (36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3). The median birth weight was 2.8 (1.3\u0026ndash;4) kg, with a mean of 2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;.6 kg. The median birth length was 45 (39\u0026ndash;52) cm, with a mean of 45.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99 cm. Within the 30\u0026ndash;35 range, the average newborn head circumference was 33.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8. Ten (12.7%) newborns had amniotic fluid stained with meconium, while sixty (75.9%) babies were cephalic presentation.\u003c/p\u003e \u003cp\u003eEffects of neonatal and maternal variables on the passage (P1) doubling time:\u003c/p\u003e \u003cp\u003eFor WJ-MSCs, the average P1 doubling time was 68 (55\u0026ndash;90) hours. When the association between P1 doubling time and maternal variables were analyzed, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC showed that P1 doubling time had a small positive correlation with gravidity (r\u0026thinsp;=\u0026thinsp;.624, P\u0026thinsp;\u0026lt;\u0026thinsp;.001), parity (r\u0026thinsp;=\u0026thinsp;.551, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.001), and a strong positive correlation with maternal age (r\u0026thinsp;=\u0026thinsp;.827, P\u0026thinsp;\u0026lt;\u0026thinsp;.001). P1 doubling time was not significantly associated with any of the neonatal variables: birth head circumference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.543), birth length (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.236), birth weight (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.735), or gestational age (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.186). There was no statistically significant difference in the median doubling time between preterm newborns (69 hours) and full term babies (67.5 hours) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.612). In addition, there was no statistically significant difference in the hours of male (66) and female (68) babies (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.612). The presence of amniotic fluid stained with meconium (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.238) or fetal mal-presentation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.502) had no discernible effect on the P1 doubling time. Double time and UC width were shown to have a statistically significant negative connection (r=-.5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMaternal and neonatal variables' effects on the yield of WJ-MSCs:\u003c/p\u003e \u003cp\u003eBy the end of P3, the mean WJ-MSCs yield was (2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;.74\u0026times;106) cell/ml [2.6 (.4\u0026ndash;3.5) \u0026times;106 cell/ml, according to the median (range). For the median (range), the viability percentage was 83.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6% [84.5 (65\u0026ndash;97)%]. UC samples were used to determine the WJ-MSCs count of full-term neonates (2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;.74 x 106 cell/ml; median\u0026thinsp;=\u0026thinsp;2.6 (.4\u0026ndash;3.5)\u0026times;106 cell/ml) and preterm newborns (2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;.76 x 106 cell/ml; median\u0026thinsp;=\u0026thinsp;2.6 (.7\u0026ndash;3.3)\u0026times;106 cell/ml). No statistically significant difference was seen in the median WJ-MSCs yield between preterm and full-term newborns (Mann-Whitney Up =\u0026thinsp;.742).\u003c/p\u003e \u003cp\u003eMaternal age and WJ-MSCs yield had a negative correlation (r=-.246, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.029) according to an analysis of maternal variables (Table\u0026nbsp;1). There was no statistically significant difference in the median WJ-MSCs yield between UC specimens obtained from women with and without gestational diabetes. In UC specimens collected from mothers with or without pre-eclampsia, WJ-MSCs yield was also similar. It is noteworthy, therefore, that there were very few pre-eclamptic or gestational diabetic individuals in the study. WJ-MSCs yield did not significantly correlate with maternal hemoglobin level prior to birth, parity, or gravidity.\u003c/p\u003e \u003cp\u003eAnalyses of the association between WJ-MSCs yield and newborn characteristics (Table\u0026nbsp;1) revealed a positive correlation between the WJ-MSCs count and birth weight and gestational age. However, it was shown that the yield of WJ-MSCs and UC width had a weakly positive correlation. A multivariate linear regression analysis revealed that the sole independent predictor of WJ-MSCs production was maternal age (Table\u0026nbsp;2). The yield of WJ-MSCs was not independently predicted by gestational age, birth weight, or cord breadth (P\u0026thinsp;=\u0026thinsp;0.330).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA promising source of MSCs for therapeutic uses is Wharton's Jelly. WJ-MSCs are easily accessible with many advantages over the BM-MSCs. Several maternal and neonatal parameters have been studied in relation to the characterization and quality of hematopoietic UC blood stem cells in the past. Although research on how maternal and neonatal variables affect the yield of WJ-MSCs is still under research investigation. For the purpose of aiding in finding and selecting the best UC donors, this study studied the effects of many maternal and fetal factors on the yield of WJ-MSCs.\u003c/p\u003e \u003cp\u003eAccording to our findings, the mother's age has a substantial impact on the yield of WJ-MSCs. This is in agreement with the findings of [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], who found that whereas UC-MSCs from elder donors show diminished differentiation potential, UC from younger donors is a reasonably abundant source of MSCs. Furthermore, compared to younger age groups, there was a significant negative correlation in the expression levels of both CD29 and CD105, according to [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It was reported that as a woman ages, her mesenchymal stem cells expressing the BIRC2 and BIRC3 genes decrease as stem cells obtained from younger women may exhibit more apoptotic resistance and a more stem cell-like quality, which may enhance their therapeutic potential and clinical usability [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA known pluripotent transcription factor, SOX2 is implicated in reprogramming, self-renewal, and maintaining the homeostasis of stem cells. The SOX2 gene is statistically considerably more expressed in WJSC in women under the age of 34 than it is in women over 34, according to [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, a statistically significant moderate negative correlation is demonstrated between the SOX2 gene expression and the maternal age. This result was clarified by [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], who found that aging in mothers is related to telomere shortening and increased susceptibility to apoptosis, and that stem cells' telomere length could not be maintained by the level of telomerase activity.\u003c/p\u003e \u003cp\u003eThese results largely stated the decline in CD105, CD73, and CD90 expression levels that they reported in their study's UC-MSCs as they were older. Furthermore, it has been previously reported how aging affects adult stem cells. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] found a decrease in adult MSCs number derived from the bone marrow with aging. According to [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], there is a decrease in the quantity, functionality, and multi-lineage differentiation characteristics of mature MSCs as they age. Additionally, the authors hypothesized that the lower differentiation capacity of the older donor's umbilical cord cells could be related to the declining functional state of the mother's older organs, which support and foster the development of umbilical cord MSC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Conversely to this finding, maternal age had no effect on the quality of WJ-MSCs, according to [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], vaginal and cesarean births had MSC cell yields that were comparable. According to [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the method of delivery had no impact on the characteristics or survivability of MSCs. To reduce the expectation of contamination, we only used specimens from Cesarean births in our investigation. We observed that the WJ-MSCs count and doubling time were not more impacted by birth order. A reduction in the number of WJ-MSCs was correlated with increasing parity, nevertheless the relationship was not statistically significant. Birth order and viability showed a moderately positive association (r\u0026thinsp;=\u0026thinsp;.119, p\u0026thinsp;=\u0026thinsp;.044), whereas parity and the amount of UC blood showed no correlation (P\u0026thinsp;=\u0026thinsp;.057) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. According to [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], WJ-MSCs obtained from younger women who gave vaginal delivery have increased expression of the BIRC2, BIRC3, and BIRC5 genes.\u003c/p\u003e \u003cp\u003ePre-eclampsia had no obvious effect on the population doubling time or WJ-MSC yield in this study. In contrast, pre-eclampsia has been shown by [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] to result in a considerable increase in cell count and reduction in the fraction of immature cells and increasing the mature cells. According to [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] compared to normal samples, the proliferation rate of early-passage gestational diabetes mellitus (DM) samples was significantly lower. Furthermore, with a mean cell density of 1.9 x 10\u003csup\u003e4\u003c/sup\u003e cells per plate in gestational DM samples as opposed to 4.1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per plate in normal samples, the population doubling rate was considerably lower in these samples after 12 days of incubation. Gestational DM-UC-MSCs did not grow above passages 6 or 7. All UC-MSCs had comparable amounts of the stem cell markers CD90, CD73, and CD105 during early stages. These results are consistent with a study conducted by [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which found that MSCs derived from Wharton Jelly of the DM group grew and proliferated at a much slower rate in all passages than MSCs isolated from a control group. The quantity of non-viable cells in the gestational DM samples significantly increased than viable cells.\u003c/p\u003e \u003cp\u003eThe WJ-MSCs count was shown to be statistically significantly impacted by gestational age in our investigation, although there was not a significant association established between gestational age and P1 doubling time. Nevertheless, there was no statistically significant difference in the median MSC output between full-term and preterm newborn when comparing them to full-term babies. In a study by [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] divided 45 babies into three groups based on their gestational ages: group A (28\u0026ndash;31 weeks), group B (32\u0026ndash;35 weeks), and group (C)\u0026thinsp;\u0026ge;\u0026thinsp;36 weeks. The authors observed a significant inverse relationship between gestational age and MSC production, even after controlling for birth weight, the percentage of small for gestational age neonates, maternal co-morbidities, and prenatal steroid usage. Similar findings were made by [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], who found that despite both samples maintained a high rate of proliferation, preterm cord samples developed more MSC in all passages than term cords. On the other hand, according to [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], the quantity of umbilical cord vein UC-MSCs from both full term and preterm patients is comparable, and both groups exhibit a high rate of proliferation. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], on the other hand, found no correlation between gestational age and MSC yield.\u003c/p\u003e \u003cp\u003eAge at gestation may possibly have an impact on the UC-MSCs' potential to modulate immunity. According to [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], preterm UC-MSCs do not have the same potent inhibitory effects on immune cell proliferation as full-term UC-MSCs. A lymphocyte co-culture experiment revealed that both full term and preterm UC-MSCs reduced peripheral blood mononuclear cell proliferation in the mixed lymphocyte response. Compared to preterm UC-MSCs, the term UC-MSCs had a stronger proliferation inhibition. The scientists explained the inhibitory difference by the preterm UC-MSCs' immature immune systems. The source, donor age, and passage number of cells can also have an impact on the immuno-modulatory qualities of MSCs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], placental weight, birth weight, and full term birth were the primary factors influencing cell proliferation. Despite the strong correlation between these parameters and gestational age, multivariate regression analysis revealed that gestational age was the only independent predictor of population doubling time (P\u0026thinsp;=\u0026thinsp;.0094). Our study found that the baby's birth weight had a substantial impact on the number of WJ-MSCs; a greater birth weight leads to increased the yield of MSCs. According to [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the yield of MSCs was not significantly affected by the mother's age or gestational age, but a greater birth weight was associated with a more yield, which is consistent with our findings. On the other hand,[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] found that lower birth weight correlates to higher SOX2 gene expression in WJ-SCs. SOX2 is a known pluripotent transcription factor that contributes to cell reprogramming and self-renewal. According to [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the MSCs output was not significantly impacted by birth weight. The author explained that his study focused on the quality of the stem cells not the number so the study finding differs from other studies that correlate the number of isolated stem cells to the birth weight [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to our findings, there was no statistically significant variation in the WJ-MSCs yields with respect to fetal gender, presentation, length at birth, or head circumference. According to [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], the mean MSCs number of female neonates was greater than that of male infants. When comparing MSCs separated from WJ from female individuals to MSCs obtained from WJ from male subjects, [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] observed that the expression of OCT4 and DNMT1 genes was considerably greater in WJ-MSCs derived from male participants. They came to the conclusion that gender significantly affects stemness genes. However, [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] found that gender did not affect the quality of WJ-MSCs.\u003c/p\u003e \u003cp\u003eWe discovered a statistically significant negative association between doubling time and UC width and a slight positive correlation between MSCs yield and UC width.\u003c/p\u003e \u003cp\u003eWe discovered that the amount of meconium in the amniotic fluid had no effect on the production of MSCs or the population doubling time. According to [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], there was little change in the expression of the mesenchymal markers CD90 and CD105 following exposure to meconium. Amniotic fluid MSCs may change in phenotype after being exposed to meconium, although they are still capable of differentiating.\u003c/p\u003e \u003cp\u003eTo sum up, Wharton's jelly MSCs are a perfect and exciting source of MSCs. Numerous factors related to mothers and newborns may impact the MSC output and should be investigated for settling the optimal selection criteria for the ideal donors. Our research showed that a higher yield of MSCs produced from Wharton's jelly correlates to a younger mother's age, increased birth weight, and longer gestational age.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.M., B.A. and Y.S. designed and supervised the work; M.R, D.A. and Y.S. performed the experiment. B.M., B.A. and M.R. performed the analysis; D.A., E.N, M.R. and Y.S. drafted the manuscript and designed the figures. B.M., B.A. and Y.S. aided in interpreting the results and worked on the manuscript. All authors discussed the results and commented on the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003e\"The data that support the findings of this study are available on request from the corresponding author, E.N.\"\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnthony, D.F. and P.G. Shiels, \u003cem\u003eExploiting paracrine mechanisms of tissue regeneration to repair damaged organs\u003c/em\u003e. Transplantation research, 2013. 2: p. 1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimran, E., et al., \u003cem\u003eExpansion and preservation of the functional activity of adult hematopoietic stem cells cultured ex vivo with a histone deacetylase inhibitor\u003c/em\u003e. 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Cold Spring Harbor Laboratory Press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, K.-R. and K.-S. Kang, \u003cem\u003eAging-related genes in mesenchymal stem cells: a mini-review\u003c/em\u003e. Gerontology, 2013. 59(6): p. 557\u0026ndash;563.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePenolazzi, L., et al., \u003cem\u003eInfluence of obstetric factors on osteogenic potential of umbilical cord-derived mesenchymal stem cells\u003c/em\u003e. Reproductive Biology and Endocrinology, 2009. 7: p. 1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRezniczek, G.A., et al., \u003cem\u003eFactors influencing yield and neuronal differentiation of mesenchymal stem cells from umbilical cord blood and matrix\u003c/em\u003e. Regenerative Medicine, 2016. 11(5): p. 465\u0026ndash;474.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Deghaither, S.Y., \u003cem\u003eImpact of maternal and neonatal factors on parameters of hematopoietic potential in umbilical cord blood\u003c/em\u003e. Saudi medical journal, 2015. 36(6): p. 704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGil-Kulik, P., et al., \u003cem\u003eIncreased expression of BIRC2, BIRC3, and BIRC5 from the IAP family in mesenchymal stem cells of the umbilical cord wharton\u0026rsquo;s jelly (WJSC) in younger women giving birth naturally.\u003c/em\u003e Oxidative medicine and cellular longevity, 2020. 2020: p. 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCervera, A., et al., \u003cem\u003eFlow cytometric assessment of hematopoietic cell subsets in cryopreserved preterm and term cord blood, influence of obstetrical parameters, and availability for transplantation\u003c/em\u003e. American journal of hematology, 2006. 81(6): p. 397\u0026ndash;410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, J., et al., \u003cem\u003eUmbilical cord mesenchymal stromal cells affected by gestational diabetes mellitus display premature aging and mitochondrial dysfunction\u003c/em\u003e. Stem Cells and Development, 2015. 24(5): p. 575\u0026ndash;586.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWajid, N., et al., \u003cem\u003eThe effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells\u003c/em\u003e. Cell and tissue banking, 2015. 16: p. 389\u0026ndash;397.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, A., et al., \u003cem\u003eDoes mesenchymal stem cell population in umbilical cord blood vary at different gestational periods?\u003c/em\u003e The Indian Journal of Pediatrics, 2013. 80: p. 375\u0026ndash;379.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRavera, S., et al., \u003cem\u003eMesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation\u003c/em\u003e. Cellular and molecular life sciences, 2018. 75: p. 889\u0026ndash;903.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbolhasani, M., et al., \u003cem\u003eImmunomodulatory properties of umbilical cord vein mesenchymal stromal cells influenced by gestational age and in vitro expansion\u003c/em\u003e. Immunology Letters, 2018. 194: p. 62\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X., et al., \u003cem\u003eTherapeutic effect of human umbilical cord mesenchymal stem cells on neonatal rat hypoxic\u0026ndash;ischemic encephalopathy\u003c/em\u003e. Journal of Neuroscience Research, 2014. 92(1): p. 35\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Q., et al., \u003cem\u003eComparative analysis of human mesenchymal stem cells from fetal-bone marrow, adipose tissue, and Warton's jelly as sources of cell immunomodulatory therapy\u003c/em\u003e. Human Vaccines \u0026amp; Immunotherapeutics, 2016. 12(1): p. 85\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvercenc-L\u0026eacute;ger, L., et al., \u003cem\u003eUmbilical cord-derived mesenchymal stromal cells: predictive obstetric factors for cell proliferation and chondrogenic differentiation\u003c/em\u003e. Stem cell research \u0026amp; therapy, 2017. 8: p. 1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazzoccoli, G., et al., \u003cem\u003eTime related variations in stem cell harvesting of umbilical cord blood\u003c/em\u003e. Scientific Reports, 2016. 6(1): p. 21404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalzano, F., et al., \u003cem\u003eLessons from human umbilical cord: Gender differences in stem cells from Wharton\u0026rsquo;s jelly\u003c/em\u003e. European Journal of Obstetrics \u0026amp; Gynecology and Reproductive Biology, 2019. 234: p. 143\u0026ndash;148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJensen, T., J. Shui, and C. Finck, \u003cem\u003eThe effect of meconium exposure on the expression and differentiation of amniotic fluid mesenchymal stem cells\u003c/em\u003e. Journal of Neonatal-Perinatal Medicine, 2017. 10(3): p. 313\u0026ndash;323.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable (1): A summary of the correlation between \u0026nbsp; neonatal and maternal variables and the yield of WJ-MSCs\u0026nbsp;\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"555\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eMaternal variables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003eCorrelation coefficient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eMother\u0026apos;s age (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e-.246*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.029*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eMother\u0026apos;s hemoglobin level (gm/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.743\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eParity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.545\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eGravidity\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eToxemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.732**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eGestational diabetes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.762**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eNeonatal variables\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eGestational age in weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.004*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.681**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eFetal presentation\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.373**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eNewborn birth weight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.015*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eThe width of Umbilical Cord (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.285\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.011*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eBirth length (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;.057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.620\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eThe head circumference (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e.130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.253\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.288288288288285%\" valign=\"top\"\u003e\n \u003cp\u003eMeconium stained amniotic fluid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.35135135135135%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.36036036036036%\" valign=\"top\"\u003e\n \u003cp\u003e.308**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eP values \u0026lt; .05*, **Mann-Whitney U test.\u003c/p\u003e\n\u003cp\u003eTable (2): The WJ-MSCs yield model using linear regression analysis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"582\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.144329896907216%\" valign=\"top\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.896907216494846%\"\u003e\n \u003cp\u003eStandardized ẞ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.95876288659794%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.144329896907216%\" valign=\"top\"\u003e\n \u003cp\u003eThe age of gestation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.896907216494846%\"\u003e\n \u003cp\u003e-.151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.95876288659794%\"\u003e\n \u003cp\u003e.563\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.144329896907216%\" valign=\"top\"\u003e\n \u003cp\u003eMaternal age\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.896907216494846%\"\u003e\n \u003cp\u003e-.249\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.95876288659794%\"\u003e\n \u003cp\u003e.027\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.144329896907216%\" valign=\"top\"\u003e\n \u003cp\u003eNeonate birth weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.896907216494846%\"\u003e\n \u003cp\u003e.302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.95876288659794%\"\u003e\n \u003cp\u003e.051\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.144329896907216%\" valign=\"top\"\u003e\n \u003cp\u003eThe width of Umbilical Cord\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.896907216494846%\"\u003e\n \u003cp\u003e.210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.95876288659794%\"\u003e\n \u003cp\u003e.330\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e(Adjusted \u003cem\u003eP\u003c/em\u003e=.003, R\u003csup\u003e2\u003c/sup\u003e=.146).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Wharton's jelly, Stem cells, Yield, Maternal- Neonatal, Flow cytometry","lastPublishedDoi":"10.21203/rs.3.rs-4437032/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4437032/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBecause Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are easily accessible, easy to isolate, and ethically acceptable, they represent a talented source of MSCs for use in regenerative medicine. Considering decisions on WJ-MSCs collection requires extensive knowledge of the factors that impact their yield. This study's objective was to evaluate the influence of parameters related to mothers and newborns on the WJ-MSCs yield. The WJ-MSCs were isolated and expanded after being taken from 79 umbilical cord samples. Population doubling time and cell proliferation were assessed. By flow cytometry analysis, WJ-MSCs were identified by positivity of CD105, CD90, and CD73 and negativity of CD45 and CD34. There was a statistically significant negative correlation between umbilical cord width and P1 doubling time. Maternal age and WJ-MSC yield were shown to be negatively correlated. Birth weight and gestational age showed a significant positive correlation between WJ-MSCs yield and neonatal variables. No significant correlations were detected between the WJ-MSCs and the mother parity, nor the neonatal sex, fetal presentation, or head circumference. The WJ-MSCs yield increases with younger maternal age, increased gestational age, and increased neonatal birth weight. 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