Stem cells derived from amniotic fluid: new potentials in regenerative medicine

Human amniotic fluid cells have been used as a diagnostic tool for the prenatal diagnosis of fetal genetic anomalies for more than 50 years. Evidence provided in the last 5 years, however, suggests that they can also harbour a therapeutic potential for human diseases, as different populations of fetal-derived stem cells have been isolated from amniotic fluid. Mesenchymal stem cells were the first to be described, which possess the higher proliferation and differentiation plasticity of adult mesenchymal stem cells and are able to differentiate towards mesodermal lineages. Amniotic fluid stem cells have more recently been isolated. They represent a novel class of pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, as they are able to differentiate into lineages representative of all three germ layers but do not form tumours when injected in vivo. These characteristics, together with the absence of ethical issues concerning their employment, suggest that stem cells present in the amniotic fluid might be promising candidates for tissue engineering and stem cell therapy of several human disorders.     

Human amniotic fluid stem cells have better potential in early second trimester of pregnancy and can be reprogramed to iPS

Objective

To study the difference of amniotic fluid stem cell potential at different gestational age.

Neurogenic cells in human amniotic fluid

OBJECTIVE: The purpose of this study was to determine whether human amniotic fluid contains cells that harbor the potential to differentiate into neurogenic cells. STUDY DESIGN: Amniotic fluid cells (uncultivated or cultivated in standard or in neurogenic differentiation medium) were analyzed for morphologic neurogenic differentiation and for expression of cluster of differentiation 133 (marker for neuronal stem cells), nestin (neuronal progenitor cells), neurofilament (neurons), the p75 common neurotrophin receptor, the brain-derived neurotrophic factor and neurotrophin-3 and cyclic nucleotide phosphodiesterase (oligodendrocytes). RESULTS: The appearance of neurogenic cells was not detected in uncultivated cells, was sporadic after cultivation in standard medium but strongly increased in neurogenic differentiation medium, and was accompanied by the induction of the expression of the analyzed marker genes. CONCLUSION: For the first time, this study provides evidence that human amniotic fluid contains cells that express markers for neuronal stem and progenitor cells, which harbor the potential to differentiate into neurogenic cells.     

Will stem cells in cord blood, amniotic fluid, bone marrow and peripheral blood soon be unnecessary in transplantation?

There are now various sources of stem cells. Those derived from blastocysts, named embryo stem (ES) cells, have attracted most attention and are highly multipotent. Human cord blood became widely used as a source of stem cells with differing properties to ES cells and their therapeutic application has grown steadily as they are stored in increasing numbers of stem cell banks. Other sources of human stem cells are derived from peripheral blood and amniotic fluid. They may arise from a common origin in epiblast. This review stresses the use of cord blood stem cells, but describes new approaches which may supersede the use of most stem cells. The advantages and disadvantages of these various classes are described in relation to potential methods involving gene conversion to change somatic cells to ES cells.     

In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

To characterize different cell populations in the human ovary, morphological and functional characteristics of cell populations collected during routine IVF procedures were studied. Cells obtained from follicular fluid grew in vitro under minimal medium conditions, without growth factor, including leukaemia-inhibiting factor. Morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial-like and also neuron-like features. Morpho-functional characteristics of fibroblast-like cells were similar to mesenchymal stem cells, and, in particular, were positive for mesenchymal stemness markers, including CD90, CD44, CD105, CD73, but negative for epithelial proteins, such as cytokeratins, CD34 and CD45 antigens. Cell proliferation activity at different times and colony-forming unit capability were evaluated, and multipotency of a subset of granulosa cells was established by in-vitro differentiation studies (e.g. osteogenic, chondrogenic and adipogenic differentiation). This study suggests that cells provided by mesenchymal plasticity can be easily isolated by waste follicular fluid, avoiding scraping of human ovaries, and cultivated in minimal conditions. Successful growth of such progenitor cells on three-dimensional cryogel scaffold provides the basis for future developments in tissue engineering. This culture system may be regarded as an experimental model in which biological behaviour is not influenced by specific growth factors.     

Amniotic membrane and amniotic cells: potential therapeutic tools to combat tissue inflammation and fibrosis?

In addition to the placenta, umbilical cord and amniotic fluid, the amniotic membrane is emerging as an immensely valuable and easily accessible source of stem and progenitor cells. This concise review will focus on the stem/progenitor cell properties of human amniotic epithelial and mesenchymal stromal cells and evaluate the effects exerted by these cells and the amniotic membrane on tissue inflammation and fibrosis.     

Endometrial reconstruction from stem cells

Adult stem cells have been identified in the highly regenerative human endometrium on the basis of their functional attributes. They can reconstruct endometrial tissue in vivo suggesting their possible use in treating disorders associated with inadequate endometrium. The identification of specific markers for endometrial mesenchymal stem cells and candidate markers for epithelial progenitor cells enables the potential use of endometrial stem/progenitor cells in reconstructing endometrial tissue in Asherman syndrome and intrauterine adhesions.     

Human Fetal Membranes: A Source of Stem Cells for Tissue Regeneration and Repair?

The ability of stem cells to differentiate into multiple cell lineages has ushered in exciting possibilities for stem cell based therapies that would be used to regenerate and repair damaged tissues and organs. Stem cells isolated from the embryo, fetus, adult and also the umbilical cord and placenta are being widely tested. Recent studies show that human fetal membranes also harbour cells with stem cell like properties. The amnion and chorion contain stromal cells that display characteristics and differentiation potential similar to that of adult, bone marrow derived mesenchymal stem cells. Amniotic epithelial cells share some of the features of pluripotent embryonic stem cells and multipotent mesenchymal stem cells and differentiate into multiple cell lineages in vitro. Amniotic epithelial cells also produce numerous substances that could augment tissue regeneration and repair. This review will focus on the stem cell like properties of stromal and epithelial cells derived from human fetal membranes and their potential use in stem cell based therapies.     

Identification of stem cell marker-positive cells by immunofluorescence in term human amnion

The placenta contains different populations of stem/progenitor cells such as mesenchymal, hematopoietic, trophoblastic and pluripotent stem cells. Although some tissue-specific stem cells are restricted to particular parts of the placenta, the localization of embryonic stem cell-like cells in term human placenta has not been determined. We have used immunofluorescence staining techniques with antibodies to pluripotent stem cell antigens, SSEA-3, SSEA-4, TRA 1-60 and TRA 1-81, and confocal microscopic analysis to identify and localize stem cells within the placenta. Stem cell marker-positive cells were found in amnion but not in choriodecidua, tissues known to contain hematopoietic and trophoblastic stem cells. Amniotic mesenchymal cells did not react with these pluripotent stem cell markers, while all amniotic epithelial cells reacted with at least one antibody. The TRA 1-60 and TRA 1-81 positive cells were solitary and present throughout the surface of amniotic membrane without a specific pattern of distribution, whereas SSEA-3 was negative and SSEA-4 was weakly positive on all amniotic epithelial cells. These data suggest that the human amnion contains stem cell-like cells at different states of differentiation. Human term amnion may be useful source of pluripotent stem cells for regenerative medicine.     

Detection and enumeration of colonic mucosal cells in amniotic fluid using a colon epithelial-specific monoclonal antibody

Since its introduction, prenatal diagnosis of chromosomal and metabolic disorder by midtrimester amniocentesis has relied upon the use of a mixture of fetal cells obtained from amniotic fluid. Little knowledge has been gained in the sorting of these cells for diagnosis of tissue-specific disorders. In an attempt to determine the contribution of fetal colonic mucosal cells to the overall amniocyte population, we used the colonic epithelial-specific monoclonal antibody (MC-Ab) 7E12H12, IgM isotype. Specimens of the small intestine, colon, buccal mucosa, kidney, urinary bladder, and umbilical cord were obtained from electively aborted normal fetuses of 12-28 weeks' gestation. All of these specimens were examined with 7E12H12 by the immunoperoxidase technique. The MC-Ab reacted with the colonic epithelial cells but not with any of the other tissues. In addition, 40 amniotic fluid samples obtained from women between 16 and 18 weeks of gestation, who underwent amniocentesis because of advanced maternal age, were tested using a fluorescent activated cell sorter. Among the amniotic fluid specimens examined, 18.4 +/- 10.3 per cent cells reacted with 7E12H12. Double immunofluorescence studies revealed that all Mc-Ab-stained cells contained secretory component, confirming that they were epithelial in origin. All fetuses whose amniotic fluid was analysed had normal karyotypes and amniotic fluid alpha-fetoprotein levels that were also normal. This study demonstrates that cell-specific Mc-Ab can be used to detect colon cells in the amniotic fluid and that colon cells contribute significant numbers in the mixture of amniotic fluid cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis

IntroductionHuman umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date.MethodsWe analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord –in situ analysis– and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence.ResultsOur results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers.ConclusionsThese results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering.     

妊娠晚期羊水间充质干细胞生物学特性研究

目的：研究妊娠晚期羊水来源的胎儿间充质干细胞（MSCs）的体外分离、培养及其生物学特性。方法：贴壁培养法培养妊娠晚期羊水中的胎儿MSCs，形成细胞集落后用细胞刮刀刮下细胞重新培养、扩增，用倒置相差显微镜观察细胞形态．通过细胞表面抗原、细胞核型分析、细胞因子检测等证实该细胞是否为胎儿MSCs，以及不同代细胞之间是否有生物学特性上的差异，并通过绘制生长曲线，比较各代细胞的生长趋势。结果：用贴壁培养法分离、培养出的细胞呈梭形，不同代胎儿MSCs表面抗原、细胞核型相同，不同羊水标本来源的胎儿MSCs均表达胚胎干细胞相关蛋白（Oct-d），第8代（P8代）细胞比P2、P5代细胞生长缓慢。结论：通过上述方法可以成功分离、培养妊娠晚期羊水来源的胎儿MSCs，不同代细胞间生物学特性基本相同。     

Differentiation of amniotic epithelial cells into various liver cell types and potential therapeutic applications

The aim of Regenerative Medicine is to replace or regenerate human cells, tissues or organs in order to restore normal function. Among all organs, the liver is endowed with remarkable regenerative capacity. Nonetheless, there are conditions in which this ability is impaired, and the use of isolated cells, including stem cells, is being considered as a possible therapeutic tool for the management of chronic hepatic disease.Placenta holds great promise for the field of regenerative medicine. It has long been used for the treatment of skin lesions and in ophthalmology, due to its ability to modulate inflammation and promote healing. More recently, cells isolated from the amniotic membrane are being considered as a possible resource for tissue regeneration, including in the context liver disease. Two cell types can be easily isolated from human amnion: epithelial cells (hAEC) and mesenchymal stromal cells (hAMSC). However only the first cell population has been demonstrated to be a possible source of proficient hepatic cells. This review will summarize current knowledge on the differentiation of hAEC into liver cells and their potential therapeutic application.     

Premature rupture of membranes and surface energy: possible role of surfactant

Thirty-one human placental membranes of various gestational ages (24 1/2 to 42 weeks) have been studied for their surface properties by use of a goniometer to observe drops of saline and olive oil applied at regular intervals to the amniotic and chorionic surfaces as they dried in air. Results show that the epithelial surfaces of the chorion and amnion and their interface are all appreciably hydrophobic. Surface energy decreased on the amniotic surface with fetal maturity while the surface energy of the chorion was significantly (137%) higher in those displaying premature rupture of the membranes than in others of comparable gestational age. This is explained on the basis that low-energy surfaces (for instance, Teflon) do not stick and provide boundary lubrication which facilitates movement. These properties are needed to avoid the buildup of local mechanical stress which could initiate rupture. It is speculated that the release agent-lubricant is surfactant derived from amniotic fluid and directly adsorbed onto the epithelial walls. On other tissues, the same surfactants have been shown to reduce surface energy, rendering them hydrophobic and imparting many desirable properties such as release, lubrication, and resistance to erosion.     

Tissue engineering of reproductive tissues and organs

Regenerative medicine and tissue engineering technology may soon offer new hope for patients with serious injuries and end-stage reproductive organ failure. Scientists are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that can restore and maintain normal function in diseased and injured reproductive tissues. In addition, the stem cell field is advancing, and new discoveries in this field will lead to new therapeutic strategies. For example, newly discovered types of stem cells have been retrieved from uterine tissues such as amniotic fluid and placental stem cells. The process of therapeutic cloning and the creation of induced pluripotent cells provide still other potential sources of stem cells for cell-based tissue engineering applications. Although stem cells are still in the research phase, some therapies arising from tissue engineering endeavors that make use of autologous adult cells have already entered the clinic. This article discusses these tissue engineering strategies for various organs in the male and female reproductive tract.     

High-efficiency retroviral transduction of fetal liver CD38-CD34++ cells: implications for in utero and ex utero gene therapy

OBJECTIVES: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. METHODS: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38-CD34++lineage- (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (Delta NGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 microg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10-21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. RESULTS: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). CONCLUSIONS: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.     

Stem cells in gynaecology

Currently, there is enormous interest in stem cells as a new treatment modality for regenerative medicine, commencing when human embryonic stem (hES) cells were first cultured from spare in vitro fertilisation-derived embryos. Emerging evidence also suggests that somatic stem cells may have greater differentiation potential. Stem cell research is now in an exciting phase of development and has the potential to dramatically influence therapeutics as hES cell derivatives and/or adult stem cells are applied to regenerative medicine or to deliver gene therapy. Human ES cells show apparently limitless proliferative potential and differentiation capacity into all tissue types. Adult stem cells are rare cells, which maintain the tissue in which they reside. The challenges facing the use of hES cells and adult stem cells in medicine are highlighted and examples of their use in laboratory studies and the clinic are given. Adult stem cells have been identified in diverse tissues, including human bone marrow, breast, prostate, brain and liver. We hypothesised that adult stem cells reside in the endometrium, a highly proliferative, cyclically regenerating tissue. Our research has demonstrated, for the first time, that human endometrium contains a small population of epithelial cells (0.22%) and stromal cells (1.25%) that exhibit stem/progenitor cell behaviour in vitro; clonogenicity. The progeny in these colonies have been characterised and growth factors supporting clonogenicity identified. The goal is to examine the role of putative endometrial stem/progenitor cells in proliferative disorders of human endometrium, such as endometriosis, adenomyosis, endometrial hyperplasia and endometrial cancer, and the action of hormone-replacement therapy on the post-menopausal endometrium.     

Endometrial stem cells

PURPOSE OF REVIEW: The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Endometrial regeneration also follows parturition and extensive resection and occurs in postmenopausal women taking estrogen replacement therapy. It is likely that adult stem/progenitor cells are responsible for this remarkable regenerative capacity. This review discusses the first published evidence for the existence of endometrial stem/progenitor cells in human and mouse endometrium. RECENT FINDINGS: Functional approaches have been used to identify candidate endometrial epithelial and stromal stem/progenitor cells, due to lack of known specific endometrial stem cell markers. Rare clonogenic cells and side population cells have been identified in human endometrial cell populations. In mouse endometrium, rare label-retaining cells have also been identified. The ability of transplanted human endometrial cells to grow endometrial tissue in animal hosts also suggests the presence of stem/progenitor cells. SUMMARY: These initial studies providing the first functional evidence for epithelial and stromal stem/progenitor cells in human and mouse endometrium lay the groundwork for further studies to characterize their stem cell properties. They also provide the impetus to discover specific markers that will enable their prospective isolation and allow their location in normal and pathological endometrium to be determined.     

Adult stem cells: their scientific interest and therapeutic future

Fascinating and provocative findings have shaken the stem cell field during these past years, which may be exploited in the future in cell replacement therapies. Continuous renewal of blood, skin, and gut cells, has long be attributed to stem cells, but it was more unexpected to identify cells that fulfil the requirements for stem-progenitor cells in many tissues with a slow turnover such as heart, kidney, muscle and brain. However, despite their lack of risk and immunological barrier, adult stem cells are yet of poor therapeutic value in many diseases, because they are available in scarce number, are poorly amplified, and loose potential with ageing, among many obstacles. Thus, the identification in adult, and more recently fetal tissues, of cells with a high proliferative capacity and multi-lineage differentiation potential has been wellcome, although their existence is still a matter of controversy. An alternative would be to activate stem cells in situ, by acting on components of the niche as recently exemplified in the hematopoetic system. Finally, as fiction meets reality, it may become possible to reprogram human adult cells in pluripotent ES cells-like, as recently demonstrated in mice.     

Derivation and characterization of progenitor stem cells from canine allantois and amniotic fluids at the third trimester of gestation

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells.