scholarly journals Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses

2017 ◽  
Vol 37 (3) ◽  
pp. 278-286 ◽  
Author(s):  
Loreta L. Campos ◽  
Fernanda C. Landim-Alvarenga ◽  
Tatícia L. Ikeda ◽  
Bianca A. Monteiro ◽  
Leandro Maia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Umbilical Cord ◽  
Amniotic Membrane ◽  
Delivery Time ◽  
Mhc Ii ◽  
Assisted Delivery ◽  
Umbilical Cord Tissue

ABSTRACT: Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.

Systematic Intracellular Biocompatibility Assessments of Superparamagnetic Iron Oxide Nanoparticles in Human Umbilical Cord Mesenchyme Stem Cells in Testifying Its Reusability for Inner Cell Tracking by MRI

2019 ◽  
Vol 15 (11) ◽  
pp. 2179-2192
Author(s):  
Yuanyuan Xie ◽  
Wei Liu ◽  
Bing Zhang ◽  
Bin Wang ◽  
Liudi Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Umbilical Cord ◽  
Cell Tracking ◽  
Superparamagnetic Iron Oxide ◽  
Human Umbilical Cord ◽  
Cell Based Therapy

Until now, there is no effective method for tracking transplanted stem cells in human. Ruicun (RC) is a new ultra-small SPIONs agent that has been approved by China Food and Drug Administration for iron supplementation but not as a stem cell tracer in clinic. In this study, we demonstrated magnetic resonance imaging-based tracking of RC-labeled human umbilical cord derived mesenchymal stem cells (MSCs) transplanted to locally injured site of rat spinal cords. We then comprehensively evaluated the safety and quality of the RC-labeled MSCs under good manufacturing practicecompliant conditions, to investigate the feasibility of SPIONs for inner tracking in stem cell-based therapy (SCT). Our results showed that RC labeling at appropriate dose (200 μg/mL) did not have evident impacts on characteristics of MSCs in vitro, demonstrating safety, non-carcinogenesis, and non-tissue inflammation in vivo. The systematic assessments of intracellular biocompatibility indicated that the RC labeled MSCs met with mandatory requirements and standards for law-regulation systems regarding SCT, facilitating translation of cell-tracking technologies to clinical trials.

In Vitro and In Vivo Evidence That Umbilical Cord Blood (UCB)-Derived CD45-/SSEA-4+/OCT-4+/CD133+/CXCR4+/Lin- Very Small Embryonic/Epiblast Like Stem Cells (VSELs) Do Not Contain Clonogenic Hematopoietic Progenitors but Are Highly Enriched in More Primitive Stem Cells - Novel View On Hierarchy of UCB Stem Cell Compartment.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 35-35 ◽  
Author(s):  
Ewa K. Zuba-Surma ◽  
Izabela Klich ◽  
Marcin Wysoczynski ◽  
Nicholas J Greco ◽  
Mary J. Laughlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

Abstract Abstract 35 Recently, we identified in umbilical cord blood (UCB) a population of very small embryonic/epiblast-like (VSEL) stem cells (Leukemia 2007;21:297–303) that are i) smaller than erythrocytes, ii) SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−, iii) respond to SDF-1 gradient and iv) possess large nuclei containing primitive euchromatin. We have demonstrated in vitro that UCB-derived VSELs did not reveal hematopoietic activity freshly after isolation, but grow hematopoietic colonies following co-culture/activation over OP-9 cells. To investigate the hierarchy of UCB-derived, CD45 negative VSELs, we employed staining with Aldefluor - detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. Subsequently, we sorted CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow sub-fractions of VSELs from UCB samples and established that freshly sorted from UCB VSELs in contrast to sorted CD45+/ CD133+/ALDHhigh and CD45+/CD133+/ALDHlow hematopoietic stem cells (HSC) did not grow colonies in vitro. However, when CD45− VSELs were activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and grew in routine methylcellulose cultures hematopoietic colonies composed of CD45+ cells. Interestingly, while CD45−/CD133+/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD45−/CD133+/ALDHlow VSELs was somehow delayed, what suggest that they needed more time to acquire hematopoietic commitment. Thus our in vitro data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD45−/CD133+/ALDHlow cells, suggesting their more primitive nature. In parallel, real time PCR analysis confirmed that while freshly isolated CD45−/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference), CD45−/CD133+/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference as compared to total UCB mononuclear cells) (Figure 1 panel A). Next hematopoietic potential of UCB-derived VSELs was tested in vivo after transplantation into NOD/SCID mice (Figure 1 panel B and C). We noticed that both CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow VSELs, give rise to human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice as assayed 4–6 weeks after transplantation. The level of human hematopoietic CD45+ cells in murine peripheral blood (PB), bone marrow (BM) and spleen (SP) were comparable for both transplanted UCB-VSELs fractions - 7.1±2.9% (PB), 23.2±0.2% (SP) and 25.2±1.0% (BM). In conclusion, our data suggest that freshly isolated very small CD45 negative UCB-VSELs are depleted from clonogeneic progenitors, however they are highly enriched for primitive HSC. Based on our in vitro and in vivo data we postulate following hierarchy of hematopoietic stem cells in UCB (from most primitive to more differentiated) i) CD45−/CD133+/ALDHlow, ii) CD45−/CD133+/ALDHhigh , iii) CD45+/CD133+/ALDHlow and iv) CD45−/CD133+/ALDHhigh. We also postulate that as we have already shown for murine BM-derived VSELs, human UCB-derived CD45 negative VSELs correspond to a population of most primitive long term repopulating HSC (LT-HSC). Of note, we also found that currently employed, routine UCB processing strategies may lead up to ∼50% unwanted loss of these small cells that are endowed with such remarkable hematopoietic activity! Disclosures: No relevant conflicts of interest to declare.

Collection, In Vitro Expansion, and Freezing of Human Stem Cells of Fetal Origin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5268-5268
Author(s):  
Eugeniusz K. Machaj ◽  
Marzena Jastrzewska ◽  
Agnieszka Gajkowska ◽  
Tomasz Oldak ◽  
Romuald Debski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Volume Reduction ◽  
Blood Samples ◽  
Fetal Stem Cells ◽  
Large Numbers ◽  
Umbilical Cord Tissue

Abstract Fetal stem cells have several therapeutical advantages when compared to their adult counterparts. Cell populations residing in “waste” tissues (cord blood, umbilical cord and placenta) may be collected without any medical or ethical contraindications concerning mother or newborn baby. Cord blood hematopoietic stem cells are routinely used for clinical transplantations, and the methods for their collection, isolation and freezing are now well standardized. We have tested the efficiency of cell separator (Sepax, Biosafe) in volume reduction of cord blood units, and in washing-out DMSO after cell thawing. Results of the volume-reduction procedure revealed that the average MNC recovery rate was 91±5%, cell viability was ≥90%, and CD34+ cell recovery rate was 92±4%. Volume reduction process eliminated 88±4.5% of red cells. In vitro clonogenic potential of cells isolated with Sepax did not differ from this of cells isolated according to the standard HES sedimentation protocol. Protocol of Sepax-based washing of thawed cells resulted of 9±4% cell loss and 10±6% decrease in cell viability, when compared to thawed non-processed cells. We conclude, that Sepax separator may be used both for cord blood volume reduction, and DMSO elimination from thawed samples. The other cord blood stem cell population, identified by us (J Cell Sci 2002, 115:2131–2138), was plastic-adherent, nonhematopoietic stem cell differentiating at least into neuropoietic progeny. Attempted optimisation of cell isolation/culture methods did not allow for successful isolation of these cells from more than approximately 20% cord blood samples - we postulate, that this cell population, when detected in mature newborn blood, is rather a remnant of cells circulating in earlier stages of fetus development. The population of cells residing in Wharton jelly of umbilical cord (CD34−, CD45−, CD29+, CD44+, CD51+, CD105+, SH-2+, SH-3+, plastic-adherent) is capable, according to published data, to differentiate into several tissues, resembling the adult mesenchymal stem cells. We were able to isolate these cells from 85% of cord collections, and to expand them for 5–10 passages in in vitro cultures in IMDM + 10% FCS media without growth factors added. We have also developed the protocol, allowing for storage of frozen umbilical cord tissue without preliminary cell isolation and expansion, allowing for labor-economical and cost-economical banking of large numbers of umbilical cord cell samples. Conclusions: Sepax application for cord blood volume reduction, or DMSO elimination from thawed cell samples, is an improvement in cord blood bank routine protocols. The low frequency of non-hematopoietic plastic-adherent stem cells in cord blood samples, collected during mature deliveries, is rather a biological phenomenon than the inadequacy of laboratory techniques, and may reduce the utility of these cells for clinical purposes. Stem cells from Wharton’s jelly of umbilical cords, if confirmed their “stemness”, can be an easy to obtain cell source for tissue engineering. Our technique of freezing-storage of the umbilical cord tissue may be useful for low-cost, time-saving banking of large numbers of fetal stem cells.

295 EQUINE AMNION-DERIVED MESENCHYMAL STEM CELLS: POSSIBLE IMPLICATION IN ENDOMETRIAL REGENERATION

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Corradetti ◽  
R. Perego ◽  
A. Meucci ◽  
D. Bizzaro ◽  
F. Cremonesi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Amniotic Membrane ◽  
Female Reproductive Tract ◽  
Endometrial Tissue ◽  
Expression Of Genes ◽  
Endometrial Regeneration

In veterinary medicine, as in human, amnion is an attractive source of mesenchymal stem cells (MSC), which poses no ethical dilemmas, allows highly efficient recovery of cells without the requirement of invasive procedures and shows immunomodulatory properties. We previously demonstrated that equine amniotic membrane-derived cells (AMC) not only exhibit specific stem cell properties with respect to expression of pluripotent (OCT-4, TRA-1-60, and SSEA-4) and adult (CD44, CD105, CD29, and CD166) stem cell markers but also possess differentiation potential in vitro and the capability to regenerate tendons in vivo after spontaneous lesions when allogeneically transplanted. Moreover, we reported evidence that at the first passages (P) in culture (until P5), AMC express MHC-class I but not MHC-class II and are well tolerated in vivo. In the present study, we further characterised AMC in vitro in order to evaluate their potential application in the treatment of endometritis in vivo. In particular, the amniotic membrane in toto and AMC have been compared to the endometrial tissue in toto and to cells isolated from endometrium for the expression of genes involved in the proliferation and differentiation of uterine MSC during early pregnancy (AbdB-like Hoxa genes) and those influencing preimplantation conceptus development (progesterone receptor, PR, and oestrogen receptors ERα and ERβ). The AMC were isolated as recently reported by Lange-Consiglio et al. (2011 Open J. Tissue Eng. Regen. Med. 4), and endometrial cells were obtained according to the protocol described by Donofrio et al. (2008 Reprod. Biol. Endocrinol. 6, 65) for bovine cells, and slightly modified for equine cells. Total RNA was extracted from both tissues and from AMC and endometrium-derived cells immediately after isolation (P0). Reverse transcription-PCR was performed according to the standard procedures, using GAPDH and HPRT1 as reference genes. Expression of HOXA9 and PR was confirmed in all samples examined, whereas mRNA for ERβ was only detected in endometrial tissue and in cells derived from it. Expression of ERα was observed only in endometrial tissue. The expression of genes crucially involved in patterning of the female reproductive tract (HOXA9 and PR) in amnion and cells derived from it suggests that this source shares similar molecular properties with endometrium. Further studies are required to explore uterine mesenchymal-like features shared by AMC (i.e. verifying the expression of Wnt7α, Wnt5α, and Wnt4α, or the presence of the more recently characterised membrane-bound intracellular progesterone receptors PGRMC1 and mPR). These preliminary results provide an intriguing indication of the possible implication of amnion-derived cells in endometrial regeneration, in particular when poor endometrial proliferation is associated with infertility or poor pregnancy outcome.

scholarly journals Regulation of sarcomagenesis by the empty spiracles homeobox genes EMX1 and EMX2

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Manuel Pedro Jimenez-García ◽  
Antonio Lucena-Cacace ◽  
Daniel Otero-Albiol ◽  
Amancio Carnero
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Neural Crest ◽  
Tumor Suppressors ◽  
Homeobox Genes ◽  
Neuronal Cell ◽  
Cell Gene Expression

AbstractThe EMX (Empty Spiracles Homeobox) genes EMX1 and EMX2 are two homeodomain gene members of the EMX family of transcription factors involved in the regulation of various biological processes, such as cell proliferation, migration, and differentiation, during brain development and neural crest migration. They play a role in the specification of positional identity, the proliferation of neural stem cells, and the differentiation of certain neuronal cell phenotypes. In general, they act as transcription factors in early embryogenesis and neuroembryogenesis from metazoans to higher vertebrates. The EMX1 and EMX2’s potential as tumor suppressor genes has been suggested in some cancers. Our work showed that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of stem cell regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES, and PROM1). EMX protein downregulation, therefore, induced the malignance and stemness of cells both in vitro and in vivo. In murine knockout (KO) models lacking Emx genes, 3MC-induced sarcomas were more aggressive and infiltrative, had a greater capacity for tumor self-renewal, and had higher stem cell gene expression and nestin expression than those in wild-type models. These results showing that EMX genes acted as stemness regulators were reproduced in different subtypes of sarcoma. Therefore, it is possible that the EMX genes could have a generalized behavior regulating proliferation of neural crest-derived progenitors. Together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-altering populations or cancer stem cells, acting as tumor suppressors in sarcoma.

scholarly journals “Empowering” Cardiac Cells via Stem Cell Derived Mitochondrial Transplantation- Does Age Matter?

2021 ◽  
Vol 22 (4) ◽  
pp. 1824
Author(s):  
Matthias Mietsch ◽  
Rabea Hinkel
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Treatment Strategies ◽  
Cardiac Cells ◽  
Aging Effects ◽  
Beneficial Effects ◽  
Micro Rnas ◽  
New Treatment

With cardiovascular diseases affecting millions of patients, new treatment strategies are urgently needed. The use of stem cell based approaches has been investigated during the last decades and promising effects have been achieved. However, the beneficial effect of stem cells has been found to being partly due to paracrine functions by alterations of their microenvironment and so an interesting field of research, the “stem- less” approaches has emerged over the last years using or altering the microenvironment, for example, via deletion of senescent cells, application of micro RNAs or by modifying the cellular energy metabolism via targeting mitochondria. Using autologous muscle-derived mitochondria for transplantations into the affected tissues has resulted in promising reports of improvements of cardiac functions in vitro and in vivo. However, since the targeted treatment group represents mainly elderly or otherwise sick patients, it is unclear whether and to what extent autologous mitochondria would exert their beneficial effects in these cases. Stem cells might represent better sources for mitochondria and could enhance the effect of mitochondrial transplantations. Therefore in this review we aim to provide an overview on aging effects of stem cells and mitochondria which might be important for mitochondrial transplantation and to give an overview on the current state in this field together with considerations worthwhile for further investigations.

scholarly journals Heterogeneity of Umbilical Cords as a Source for Mesenchymal Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
O. O. Maslova ◽  
N. S. Shuvalova ◽  
O. M. Sukhorada ◽  
S. M. Zhukova ◽  
O. G. Deryabina ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Umbilical Cord ◽  
Optimal Conditions ◽  
Umbilical Cord Tissue ◽  
Umbilical Cords

The object of the paper is to show the heterogeneity of 300 cord samples processed in the current research. The differences in effectiveness of mesenchymal stem cell (MSC) isolation are shown. Moreover, the recommendations for choosing the method of MSC isolation depending on the value of stromal-vascular rate are given. The data can be useful for selecting the optimal conditions to obtain MSC and for further cryopreservation of umbilical cord tissue.

scholarly journals Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Stimulated by Hypoxia Promote Angiogenesis Both In Vitro and In Vivo

2012 ◽  
Vol 21 (18) ◽  
pp. 3289-3297 ◽  
Author(s):  
Hong-Chao Zhang ◽  
Xin-Bin Liu ◽  
Shu Huang ◽  
Xiao-Yun Bi ◽  
Heng-Xiang Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Human Umbilical Cord

scholarly journals Xenogenic Implantation of Equine Synovial Fluid-Derived Mesenchymal Stem Cells Leads to Articular Cartilage Regeneration

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Mohammed Zayed ◽  
Steven Newby ◽  
Nabil Misk ◽  
Robert Donnell ◽  
Madhu Dhar
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Articular Cartilage ◽  
Synovial Fluid ◽  
Cartilage Repair ◽  
Cartilage Regeneration ◽  
Large Animal

Horses are widely used as large animal preclinical models for cartilage repair studies, and hence, there is an interest in using equine synovial fluid-derived mesenchymal stem cells (SFMSCs) in research and clinical applications. Since, we have previously reported that similar to bone marrow-derived MSCs (BMMSCs), SFMSCs may also exhibit donor-to-donor variations in their stem cell properties; the current study was carried out as a proof-of-concept study, to compare the in vivo potential of equine BMMSCs and SFMSCs in articular cartilage repair. MSCs from these two sources were isolated from the same equine donor. In vitro analyses confirmed a significant increase in COMP expression in SFMSCs at day 14. The cells were then encapsulated in neutral agarose scaffold constructs and were implanted into two mm diameter full-thickness articular cartilage defect in trochlear grooves of the rat femur. MSCs were fluorescently labeled, and one week after treatment, the knee joints were evaluated for the presence of MSCs to the injured site and at 12 weeks were evaluated macroscopically, histologically, and then by immunofluorescence for healing of the defect. The macroscopic and histological evaluations showed better healing of the articular cartilage in the MSCs’ treated knee than in the control. Interestingly, SFMSC-treated knees showed a significantly higher Col II expression, suggesting the presence of hyaline cartilage in the healed defect. Data suggests that equine SFMSCs may be a viable option for treating osteochondral defects; however, their stem cell properties require prior testing before application.

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