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Schematic overview of the procedure used to collect large volumes of term amniotic fluid using a closed catheter-based system, followed by MNC isolation and cell culture
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Background
Mesenchymal stromal cells (MSCs) are currently being evaluated in numerous pre-clinical and clinical cell-based therapy studies. Furthermore, there is an increasing interest in exploring alternative uses of these cells in disease modelling, pharmaceutical screening, and regenerative medicine by applying reprogramming technologies. Howeve...
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One of the key issues hampering the development of effective treatments for prostate cancer is the lack of suitable, tractable, and patient-specific in vitro models that accurately recapitulate this disease. In this review, we address the challenges of using primary cultures and patient-derived xenografts to study prostate cancer. We describe emerg...
Citations
... AFSCs can be differentiated into neural, renal, hematopoietic, adipogenic, myogenic, endothelial, chondrogenic, osteogenic, epithelial, and hepatic cell types [17,39,[93][94][95]. AF-MSCs have been developed upon neural, adipogenic, and osteogenic lineages [49,50,96]. The differentiation potential of AM-MSCs includes the development into neural, myogenic, endothelial and hepatic cell types [51,52,95,97] and AECs differentiate into neural, adipogenic, myogenic, osteogenic, and hepatic cells [53,67,70]. ...
Due to the limited accessibility of the in vivo situation, the scarcity of the human tissue, legal constraints, and ethical considerations, the underlying molecular mechanisms of disorders, such as preeclampsia, the pathological consequences of fetomaternal microchimerism, or infertility, are still not fully understood. And although substantial progress has already been made, the therapeutic strategies for reproductive system diseases are still facing limitations. In the recent years, it became more and more evident that stem cells are powerful tools for basic research in human reproduction and stem cell-based approaches moved into the center of endeavors to establish new clinical concepts. Multipotent fetal stem cells derived from the amniotic fluid, amniotic membrane, chorion leave, Wharton´s jelly, or placenta came to the fore because they are easy to acquire, are not associated with ethical concerns or covered by strict legal restrictions, and can be banked for autologous utilization later in life. Compared to adult stem cells, they exhibit a significantly higher differentiation potential and are much easier to propagate in vitro. Compared to pluripotent stem cells, they harbor less mutations, are not tumorigenic, and exhibit low immunogenicity. Studies on multipotent fetal stem cells can be invaluable to gain knowledge on the development of dysfunctional fetal cell types, to characterize the fetal stem cells migrating into the body of a pregnant woman in the context of fetomaternal microchimerism, and to obtain a more comprehensive picture of germ cell development in the course of in vitro differentiation experiments. The in vivo transplantation of fetal stem cells or their paracrine factors can mediate therapeutic effects in preeclampsia and can restore reproductive organ functions. Together with the use of fetal stem cell-derived gametes, such strategies could once help individuals, who do not develop functional gametes, to conceive genetically related children. Although there is still a long way to go, these developments regarding the usage of multipotent fetal stem cells in the clinic should continuously be accompanied by a wide and detailed ethical discussion.
... To be identified as MSCs, cells need to fulfill certain criteria proposed by the International Society for Cellular Therapy, including expression and absence of certain surface markers [76]. Scientists are now able to isolate human MSCs from many tissues including bone marrow (BM-MSCs) [77,78], adipose tissue [77,79], umbilical cord (UC-MSCs) [77,80], amniotic fluid [81][82][83][84], Wharton's jelly [85][86][87], skin [88,89], and dental tissues [90][91][92]. MSCs have been widely used to treat SCI in animal and human clinical trials. ...
Spinal cord injury (SCI) is a devastating condition that enormously affects an individual’s health and quality of life. Neurogenic lower urinary tract dysfunction (NLUTD) is one of the most important sequelae induced by SCI, causing complications including urinary tract infection, renal function deterioration, urinary incontinence, and voiding dysfunction. Current therapeutic methods for SCI-induced NLUTD mainly target on the urinary bladder, but the outcomes are still far from satisfactory. Stem cell therapy has gained increasing attention for years for its ability to rescue the injured spinal cord directly. Stem cell differentiation and their paracrine effects, including exosomes, are the proposed mechanisms to enhance the recovery from SCI. Several animal studies have demonstrated improvement in bladder function using mesenchymal stem cells (MSCs) and neural stem cells (NSCs). Human clinical trials also provide promising results in urodynamic parameters after MSC therapy. However, there is still uncertainty about the ideal treatment window and application protocol for stem cell therapy. Besides, data on the therapeutic effects regarding NSCs and stem cell-derived exosomes in SCI-related NLUTD are scarce. Therefore, there is a pressing need for further well-designed human clinical trials to translate the stem cell therapy into a formal therapeutic option for SCI-induced NLUTD.
... For instance, PL-MSCs were superior in cell proliferation, survival, differentiation and immunomodulatory potentials compared to BM-MSCs (10). They possessed lineage differentiation capabilities beyond mesoderm such as myogenesis and neurogenesis when induced with respective media (11). Compared to ASCs, lesser risk in stimulating an allogeneic response after the administration of UC-MSCs as there was no increase in human leukocyte antigen-A, B, C (HLA-ABC) and HLA-DR expression after interferon-gamma stimulation (3,12). ...
... One of the major setbacks contributing to the wide-ranging success rate between 10% and 90% in culturing UCB-MSCs is inconsistency in the isolation process (38,42). Many researchers as well by some researchers (11,31). Moraghebi et al. (11) had demonstrated the feasibility of collecting a substantial volume of term amniotic fluid using a siphoning catheter-based system. ...
... Epithelioid-like cell population showed a slower proliferation rate than fibroblasticlike cells and could be cultured for long-term in vitro expansion. Reprograming these cells into hematopoietic and neural cell lineages is feasible (11). Thus, full-term AF could also be a promising source of mesenchymal stem cells. ...
Mesenchymal stromal/stem cells (MSCs) derived from perinatal tissues have become indispensable sources for clinical applications due to their superior properties, ease of accessibility and minimal ethical concerns. MSCs isolated from different placenta (PL) and umbilical cord (UC) compartments exhibit great potential for stem cell-based therapies. However, their biological activities could vary due to tissue origins and differences in differentiation potentials. This review provides an overview of MSCs derived from various compartments of perinatal tissues, their characteristics and current isolation methods. Factors affecting the yield and purity of MSCs are also discussed as they are important to ensure consistent and unlimited supply for regenerative medicine and tissue engineering.
... 10 In this study, we isolated and characterized the CD117 + cells (AFSCs) from Amniotic fluid, the amniotic fluid were collected during C-section, which were usually discarded at time of birth. 11 A comparison with other studies showed that CD117 + cells can be isolated from 2 nd trimester also and isolation of CD117 + cells from amniotic fluid (less than 1% of total amniotic fluid cells) is also carried various protocols as like via magnetic cell sorting or flow cytometry. 12 Amniotic fluid contain heterogeneous population of cells but among all AFSCs have both recompenses over adult and embryonic stem cells like pluripotent action, immunomodulatory properties and high plasticity that can be a rich source off future therapeutic uses. ...
Objective:
The study aimed at isolation of CD117+ stem cells from amniotic fluid samples followed by their invitro differentiation towards nephron progenitors that can be potentially used for regenerative medicine studies and to further understand pathways involved in renal pathogenesis.
Methods:
This experimental study was conducted at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences, OJHA Campus Karachi from November 2019 to December 2020. After patient consent, a Pfannenstiel incision was performed by the gynecologist through abdominal and uterine muscles without cutting into Amniotic Membrane. Using a needle of 5CC syringe connected to sterile Redivac bottle, a blunt end insertion was passed through the membrane and the amniotic fluid was aseptically sucked into Redivac bottle, the ice bag was used for transporting amniotic fluid from hospital to the lab and samples were processed within 60 minutes after collection. Amniotic fluid was centrifuged at 4º C for 20 minutes at 1400xg. After centrifugation the cell pellet was treated for analysis of CD117+ cells using flowcytometry, once small percentage of CD117+ cells were identified the cells were prepared for differentiation and that was carried out using specific growth factors including BMP4, BMP7, FGF2, and retinoic acid, providing the niche to the stem cells for differentiation towards nephron progenitors which was confirmed by protein expression of Wilms Tumor-1 (WT1) using immunofluorescence analysis. The sample size for this invitro work was n=3.
Results:
We successfully isolated small percentage of CD117+ cells in amniotic fluid followed by in vitro expansion and differentiation towards nephron progenitor cells (NPCs) using well defined media and growth factors, initially differentiated cells were spindle shaped and showed fibroblastic appearance later at stage of nephron progenitors it attained the shape of rounded big clusters, differentiated cells stained positive for WT1 and negative for cluster of differentiation (CD117). Therefore, confirming the successful isolation and differentiation of amniotic fluid stem cells towards nephron progenitors.
Conclusion:
To the best of our knowledge this is the first study from the country on the use of Amniotic fluid stem cells and their differentiation towards nephron progenitors that can be used as substitution source of cell therapy for exploration of renal diseases at cellular and molecular level and potential regenerative medicine applications.
... "PMSCs" is usually used as a general term including all the mesenchymal stem cells (MSCs) isolated from different sites of the placenta, such as the chorionic membrane (CMMSCs) (Jaramillo-Ferrada et al., 2012), the chorionic villi (CVMSCs) (Makhoul et al., 2013), chorionic cotyledons or intervillous space (CIVMSCs) Mathew et al., 2017), chorionic plate (CPMSCs) (Kim et al., 2011), the chorionic trophoblastic cells (Knöfler et al., 2019), the amniotic membrane (AMMSCs) (Tamagawa et al., 2004), the amniotic fluid (AFMSCs) (Moraghebi et al., 2017), the umbilical cord (UCMSCs) , and the decidua basalis (DBMSCs) (Abumaree et al., 2017). Though derived from different parts in the placenta, all these cells conform to the definition of MSCs according to the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006). ...
The well-developed placentation is fundamental for the reproductive pregnancy while the defective placental development is the pathogenetic basis of preeclampsia (PE), a dangerous complication of pregnancy comprising the leading causes of maternal and perinatal morbidity and mortality. Placenta-derived mesenchymal stem cells (PMSCs) are a group of multipotent stem cells that own a potent capacity of differentiating into constitutive cells of vessel walls. Additionally, with the paracrine secretion of various factors, PMSCs inextricably link and interact with other component cells in the placenta, collectively improving the placental vasculature, uterine spiral artery remolding, and uteroplacental interface immunoregulation. Recent studies have further indicated that preeclamptic PMSCs, closely implicated in the abnormal crosstalk between other ambient cells, disturb the homeostasis and development in the placenta. Nevertheless, PMSCs transplantation or PMSCs exosome therapies tend to improve the placental vascular network and trophoblastic functions in the PE model, suggesting PMSCs may be a novel and putative therapeutic strategy for PE. Herein, we provide an overview of the multifaceted contributions of PMSCs in early placental development. Thereinto, the intensive interactions between PMSCs and other component cells in the placenta were particularly highlighted and further extended to the implications in the pathogenesis and therapeutic strategies of PE.
... Phenotypic analysis confirms the expression of various MSCs-related surface markers. Most MSCs are positive for cluster of differentiation (CD)73 (5' nucleotidase) [45][46][47][48][49][50][51], CD90 (Thy 1 membrane glycoprotein) [48][49][50][51][52][53][54], CD105 (endoglin) [32,50,51,[55][56][57], CD44 (hyaluronan receptor) [48,49,58,59], and lack expressions for CD34 (hematopoietic progenitor cell antigen) [32,55,56,60,61], CD14 (myeloid cell-specific leucine-rich glycoprotein) [60][61][62][63], CD45 (protein tyrosine-phosphatase) [64][65][66][67][68], and HLA-DR (human leukocyte antigens class II DR) [62,68,69]. Individual markers include CD146 (S-endo1, melanoma cell adhesion molecule, Muc18, or glycerin) [65][66][67]70], CD29 (integrin β 1) [45-47, 52, 53, 64-66], CD49e (integrin α 5), CD54 (intercellular adhesion molecule 1), CD106 (vascular cell adhesion molecule) [63], CD146 (melanoma cell adhesion molecule) [32,55,56,67,70], CD166 (activated leukocyte cell adhesion molecule) [63,67], CD271 (low-affinity nerve growth factor receptor) [32,46,47,55,56,[65][66][67], SSEA-4 (stage-specific embryonic antigen-4) [45], Notch 1 (neurogenic locus notch homologue protein 1), HLA-ABC (human leukocyte antigens, histocompatibility complex class I molecules) [71,72], and Stro 1 (stromal antigen 1) [68]. ...
Osteoarthritis, as a degenerative disease, is a common problem and results in high socioeconomic costs and rates of disability. The most commonly affected joint is the knee and characterized by progressive destruction of articular cartilage, loss of extracellular matrix, and progressive inflammation. Mesenchymal stromal cell (MSC)-based therapy has been explored as a new regenerative treatment for knee osteoarthritis in recent years. However, the detailed functions of MSC-based therapy and related mechanism, especially of cartilage regeneration, have not been explained. Hence, this review summarized how to choose, authenticate, and culture different origins of MSCs and derived exosomes. Moreover, clinical application and the latest mechanistical findings of MSC-based therapy in cartilage regeneration were also demonstrated.
... 12 Phase contrast microscopy revealed that, first AFSCs were appeared as spindle shaped and later it showed as round clusters (resembling NPCs) as mentioned in various studies. [13][14][15][16][17][18] In present study, confirmation of nephron progenitor cells was done by WT1 antibody which is highly expressive in NPCs, whereas in prior studies, SIX2 was also expressed along with WT1. 18 Immunofluorescence assay was used for the confirmation of expression of WT1 marker. ...
Background: The incidence of kidney disease is prevailing worldwide and there is an urgent requirement for regenerative techniques such as stem cells. Objective: To identify nephron progenitor cells from transcriptional factor WT1. Study Design: Experimental analytical study Place and Duration of Study: Dow Research Institute of Biotechnology & Biomedical Sciences, Dow International Medical College, Karachi from 1st January 2019 to 31stDecember 2019 Methodology: 40 ml of amniotic fluid was extracted from 10 full term women at the time of elective caesarean. Using cell culturing, inverted phase contrast microscopy and flow cytometry techniques in addition to immune-florescence the nephron progenitor cells were identified. Results: The mean age of women was 30.3±0.4 years. Out of total 10 million WT1 expressed in three samples 1.4 million nephron progenitor cells were identified. Conclusion: Identification of nephron progenitor cells is feasible procedure for designing stem cell lines through amniotic fluid. Key words: Progenitor cell, Amniotic fluid, Stem cell initiator
... The expression of CD105 was detectable, but, significantly lower than observed in other MSC lineages. The expression of Oct-4 was significantly increased in the freshly obtained samples in comparison to in vitro cultured cells [212]. Moraghebi et al. have also reprogrammed the term hAF-MSC into the pluripotent stem cell with a similar expression of Oct-4 and NANOG to human embryonic stem cell lines. ...
... Moraghebi et al. have also reprogrammed the term hAF-MSC into the pluripotent stem cell with a similar expression of Oct-4 and NANOG to human embryonic stem cell lines. The reprogrammed cells had the potential to form teratomas and differentiate into hematopoietic and neural cell lineages [212]. ...
Mesenchymal stem/stromal cells (MSCs) are currently one of the most extensively researched fields due to their promising opportunity for use in regenerative medicine. There are many sources of MSCs, of which cells of perinatal origin appear to be an invaluable pool. Compared to embryonic stem cells, they are devoid of ethical conflicts because they are derived from tissues surrounding the fetus and can be safely recovered from medical waste after delivery. Additionally, perinatal MSCs exhibit better self-renewal and differentiation properties than those derived from adult tissues. It is important to consider the anatomy of perinatal tissues and the general description of MSCs, including their isolation, differentiation, and characterization of different types of perinatal MSCs from both animals and humans (placenta, umbilical cord, amniotic fluid). Ultimately, signaling pathways are essential to consider regarding the clinical applications of MSCs. It is important to consider the origin of these cells, referring to the anatomical structure of the organs of origin, when describing the general and specific characteristics of the different types of MSCs as well as the pathways involved in differentiation.
... AFMSCs into pluripotent stem cells, resulting in cells endowed with broad differentiation potential toward both mesodermal and ectodermal cell lineages. 12 When added to the medium as a peptide, RANKL, a cell surface protein of BM stromal cells, 13 promotes osteoblast precursor cell proliferation. 14 An adequate concentration of melatonin in the culture medium promotes the proliferation of pancreatic stem cells. ...
... 16 Moreover, full-term AFMSCs show higher correlation of transcriptome with fetal BM-MSCs than ESCs, 17 and their transcriptional expression profile is more similar to those of BM-and adipose-derived MSCs than to those of non-MSC endothelial cells and BM-MSCs derived osteoblast cells. 12 The transcriptome has also been used to explore the cell fate determination. ...
Amniotic fluid-derived mesenchymal stromal cells (AFMSCs) present different features, depending on the isolation timing and culture conditions. The lack of uniform experimental standards hinders the comparison of results from different studies on AFMSCs. Moreover, understanding the molecular mechanisms that underlie the features of AFMSCs isolated at different embryonic developmental stages might allow the obtention of more viable and highly proliferative AFMSCs through genetic modification. We isolated AFMSCs from pregnant rats at embryonic day (E)12, E15, E18, and E21 and compared their cell proliferation capacity and transcriptome. The cell counting kit-8 assay and RNA sequencing revealed that E12 and E15 AFMSCs showed different characteristics from E18 and E21 AFMSCs. Therefore, AFMSCs were divided into two groups: early (E12 and E15) and late (E18 and E21) pregnancy-stage groups. Next, we screened the gene/microRNA pair Abca4/miR-351-3p that was related to cell proliferation. Abca4 knockdown/overexpression suggested that this gene represses the proliferation of AFMSCs, which is a newly discovered function of this gene. Finally, dual luciferase reporter gene assays confirmed that miR-351-3p targeted the coding sequence of Abca4 and regulated AFMSC proliferation. miR-351-3p promotes AFMSC proliferation via targeting the coding sequence of Abca4. Our findings provide a molecular foundation for further research for obtaining AFMSCs with a higher proliferation capacity.
... In addition, the yield of MSCs from UCB is extremely low, and the isolation of UCBMSCs is not guaranteed as UCMSCs, while UCMSCs also exhibit a great advantage in terms of proliferation. [320][321][322] Amniotic fluid is another potential MSCs source [323][324][325] ; however, due to the invasive procedure, limited availability, and ethical concern, the yielded amniotic fluid MSCs (AFMSCs) will not be further discussed in this chapter, as there are no superior clinical benefits to use AFMSCs than to use aforementioned fetal MSCs based on the current understanding. ...
A plethora of both acute and chronic conditions, including traumatic, degenerative, malignant, or congenital disorders, commonly induce bone disorders often associated with severe persisting pain and limited mobility. Over 1 million surgical procedures involving bone excision, bone grafting, and fracture repair are performed each year in the U.S. alone, resulting in immense levels of public health challenges and corresponding financial burdens. Unfortunately, the innate self‐healing capacity of bone is often inadequate for larger defects over a critical size. Moreover, as direct transplantation of committed osteoblasts is hindered by deficient cell availability, limited cell spreading, and poor survivability, an urgent need for novel cell sources for bone regeneration is concurrent. Thanks to the development in stem cell biology and cell reprogramming technology, many multipotent and pluripotent cells that manifest promising osteogenic potential are considered the regenerative remedy for bone defects. Considering these cells' investigation is still in its relative infancy, each of them offers their own particular challenges that must be conquered before the large‐scale clinical application. A plethora of both acute and chronic conditions, including traumatic, degenerative, malignant, and congenital varieties, often play key roles in reducing the quality of life for many people. This is particularly true in the case of critical‐size defects where the innate self‐healing capacity of bone is inadequate for a reunion. To date, a diversity of novel multipotent/pluripotent cell sources are regarded as regenerative medicine, particularly for bone regeneration, in virtue of continued worldwide collaboration. Although their potential is irrefutable, each of the cell sources mentioned has its own drawbacks, which must be entirely understood and overcome before they are released for human clinical application.
