Human Amniotic Fluid Cells Research Articles

Raman spectroscopy analysis of human amniotic fluid cells from fetuses with myelomeningocele

Prenatal surgery for the treatment of spina bifida (myelomeningocele, MMC) significantly enhances the neurological prognosis of the patient. To ensure better protection of the spinal cord by large defects, the application of skin grafts produced with cells gained from the amniotic fluid is presently studied. In order to determine the most appropriate cells for this purpose, we tried to shed light on the extremely complex amniotic fluid cellular composition in healthy and MMC pregnancies. We exploited the potential of micro-Raman spectroscopy to analyse and characterize human amniotic fluid cells in total and putative (cKit/CD117-positive) stem cells of fetuses with MMC in comparison with amniotic fluid cells from healthy individuals, human fetal dermal fibroblasts and adult adipose derived stem cells. We found that (i) the differences between healthy and MMC amniocytes can be attributed to specific spectral regions involving collagen, lipids, sugars, tryptophan, aspartate, glutamate, and carotenoids, (ii) MMC amniotic fluid contains two particular cell populations which are absent or reduced in normal pregnancies, (iii) the cKit-negative healthy amniocyte subpopulation shares molecular features with human fetal fibroblasts. On the one hand we demonstrate a different amniotic fluid cellular composition in healthy and MMC pregnancies, on the other our work confirms micro-Raman spectroscopy to be a valuable tool for discriminating cell populations in unknown mixtures of cells.

Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content

Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics; however, they are a heterogeneous population that ranges from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit+. Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis. In particular, transcriptome profiling identified changes in cellular distribution among the different clusters that correlated with significant differential expression in pathways related to stemness, proliferation, and cell cycle checkpoints. These differences were validated by RT-PCR, immunofluorescence, WB, and cell cycle assays. Interestingly, the two populations produced secretomes with different immune-modulating and pro-regenerative potentials. Indeed, the presence of TGFβ, HGF, IDO was higher in EVs deriving from c-Kit+ cells, unlike IL-6. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting c-Kit positive fractions with higher potential in regenerative medicine applications.

Comparison of chromosomal instability of human amniocytes in primary and long-term cultures in AmnioMAX II and DMEM media: A cross-sectional study.

BackgroundThe genomic stability of stem cells to be used in cell therapy and other clinical applications is absolutely critical. In this regard, the relationship between in vitro expansion and the chromosomal instability (CIN), especially in human amniotic fluid cells (hAFCs) has not yet been completely elucidated.ObjectiveTo investigate the CIN of hAFCs in primary and long-term cultures and two different culture mediums.Materials and Methods After completing prenatal genetic diagnoses (PND) using karyotype technique and chromosomal analysis, a total of 15 samples of hAFCs from 650 samples were randomly selected and cultured in two different mediums as AmnioMAX II and DMEM. Then, proliferative cells were fixed on the slide to be used in standard chromosome G-banding analysis. Also, the senescent cells were screened for aneuploidy considering 8 chromosomes by FISH technique using two probe sets including PID I (X-13-18-21) & PID II (Y-15-16-22).Results Karyotype and interphase fluorescence in situ hybridization (iFISH) results from 650 patients who were referred for prenatal genetic diagnosis showed that only 6 out of them had culture- derived CIN as polyploidy, including mosaic diploid-triploid and diploid-tetraploid. Moreover, the investigation of aneuploidies in senesced hAFCs demonstrated the rate of total chromosomal abnormalities as 4.3% and 9.9% in AmnioMAX- and DMEM-cultured hAFCs, respectively.ConclusionhAFCs showed a low rate of CIN in two AmnioMAX II and DMEM mediums and also in the proliferative and senescent phases. Therefore, they could be considered as an attractive stem cell source with therapeutic potential in regenerative medicine.

The effects of human platelet lysate versus commercial endothelial growth medium on the endothelial differentiation potential of human amniotic fluid mesenchymal stem cells

To differentiate stem cells into endothelial cells, vascular endothelia growth factors (VEGF) serve as the major signal for stimulating the cells. However, there are other cytokines or growth factors associated with endothelial cell development and differentiation. Human platelet lysate (hPL) has been a promising reagent in cell-based therapy since it is considered as a source of bioactive molecules and growth factors. The aim of this study was to investigate the in vitro differentiation of human amniotic fluid mesenchymal stem cells (hAF-MSCs) into endothelial-like cells under hPL together with VEGF or endothelial cell growth medium 2 (EGM-2), a commercially induced medium. In this study, hAF-MSCs were isolated from human amniotic fluid cells (hAFCs) using the direct adherence method. The cells expressed CD44, CD73, CD90, and HLA-ABC at high levels and expressed Oct-4 (octamer-binding transcription factor 4) at low levels. The cells were negative for CD31, CD34, CD45, CD105 and HLA-DR. This study found that hAF-MSCs induced with hPL and VEGF had the ability to differentiate into endothelial-like cells by presenting endothelial specific markers (vWF, VEGFR2 and eNOS), forming a network-like structure on Matrigel, and producing nitric oxide (NO). This outcome was similar to those of experiments involving EGM-2 induced cells. The present findings indicate that hPL + VEGF can induce hAF-MSCs to express endothelial cell characteristics. Our findings represent an important step forward in the development of a clinically compliant process for the production of endothelial cell-derived hAF-MSCs, and their subsequent testing in future clinical trials.

Investigating the expression of pluripotency-related genes in human amniotic fluid cells: A semi-quantitative comparison between different subpopulations, from primary to cultured amniocytes

Various classifications have been proposed for human amniotic subpopulations, including classification of spindle-shaped (SS) and round-shaped (RS) cells, as well as the more referred triple-category of epithelioid (E-type) cells, amniotic fluid-specific (AF-type) cells and fibroblastoid (F-type) cells. The present study aims to investigate these amniotic subpopulations regarding the expression of some stem cell markers, including OCT4, NANOG, SOX2, C-KIT (CD117), C-MYC, KLF4, and THY1 (CD90). Flow cytometry was performed to characterize the isolated clonal subpopulations for a hematopoietic and a mesenchymal marker using PE-CD31 and FITC-CD90, respectively. A semi-quantitative RT-PCR analysis was carried out on the isolates in the second half of their lifespan when the cells were at the stationary phase of the growth curve. Characterization of isolated cells demonstrated that all clones including both epithelioid and fibroblastoid cells, had mesenchymal, not hematopoietic, lineage. RT-PCR analysis also revealed a higher expression of the target genes in epithelioid cells. Furthermore, the expression pattern of the genes and their correlations were remarkably different between primary- and long term-cultured amniocytes. Taken together, our results showed that the primary-cultured cells express the stemness genes equally, whereas the long term-cultured amniocytes exhibited a highly variable manner in the expression pattern of the genes. Diverse derivation site of amniocytes and individual genetic background can potentially explain the observed variation in the expression level of the target genes. These can also explain why amniocytes differ in many respects observed in our study, including survival rate, plastic adhesion, and growth characteristics.

Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells

Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells

Human amniotic fluid stem cells have a unique potential to accelerate cutaneous wound healing with reduced fibrotic scarring like a fetus.

Adult wound healing can result in fibrotic scarring (FS) characterized by excess expression of myofibroblasts and increased type I/type III collagen expression. In contrast, fetal wound healing results in complete regeneration without FS, and the mechanism remains unclear. Amniotic fluid cells could contribute to scar-free wound healing, but the effects of human amniotic fluid cells are not well characterized. Here, we determined the effect of human amniotic fluid stem cells (hAFS) on FS during wound healing. Human amniotic fluid was obtained by amniocentesis at 15-17 weeks of gestation. CD117-positive cells were isolated and defined as hAFS. hAFS (1 × 106) suspended in PBS or cell-free PBS were injected around wounds created in the dorsal region of BALB/c mice. Wound size was macroscopically measured, and re-epithelialization in the epidermis, granulation tissue area in the dermis and collagen contents in the regenerated wound were histologically analyzed. The ability of hAFS to engraft in the wound was assessed by tracking hAFS labeled with PKH-26. hAFS fulfilled the minimal criteria for mesenchymal stem cells. hAFS injection into the wound accelerated wound closure via enhancement of re-epithelialization with less FS. The process was characterized by lower numbers of myofibroblasts and higher expression of type III collagen. Finally, transplanted hAFS were clearly observed in the dermis until day 7 implying that hAFS worked in a paracrine manner. hAFS can function in a paracrine manner to accelerate cutaneous wound healing, producing less FS, a process resembling fetal wound healing.

P53 Is Active in Human Amniotic Fluid Stem Cells.

Despite increasing interest in human amniotic fluid cells, very little is known about the regulation and function of p53 in this cell type. In this study, we show that undifferentiated human amniotic fluid cells express p53, yet at lower levels than in cancer cells. The p53 protein in amniotic fluid cells is mainly localized in the nuclei, however, its antiproliferative activity is compromised in these cells. Igf2, a maternal imprinted gene, and c-jun, a proto-oncogene, are regulated by p53 in these cells. DNA damage leads to an increase in p53 abundance in human amniotic fluid cells and to transcriptional activation of its target genes. Interestingly, cell differentiation toward the neural lineage leads to p53 induction as differentiation progresses.

Cardiomyocytes Derived from Human CardiopoieticAmniotic Fluids

Human amniotic fluid (hAF) cells share characteristics of both embryonic and adult stem cells. They proliferate rapidly and can differentiate into cells of all embryonic germ layers but do not form teratomas. Embryoid-bodies obtained from hAF have cardiac differentiation potential, but terminal differentiation to cardiomyocytes (CMs) has not yet been described. Our purpose was to promote cardiac differentiation in hAFcells. Cells were exposed to inducing factors for up to 15 days. Only the subset of hAF cells expressing the multipotency markers SSEA4, OCT4 and CD90 (CardiopoieticAF cells) responded to the differentiation process by increasing the expression of the cardiac transcription factors Nkx2.5 and GATA4, sarcomeric proteins (cTnT, α-MHC, α-SA), Connexin43 and atrial and ventricular markers. Furthermore, differentiated cells were positive for the calcium pumps CACNA1C and SERCA2a, with approximately 30% of CardiopoieticAF-derived CM-like cells responding to caffeine or adrenergic stimulation. Some spontaneous rare beating foci were also observed. In conclusion, we demonstrated that CardiopoieticAF cells might differentiate toward the cardiac lineage giving rise to CM-like cells characterized by several cardiac-specific molecular, structural, and functional properties.

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.

Establishment of a human trisomy 18 induced pluripotent stem cell line from amniotic fluid cells.

Trisomy 18 (18T) is the second most common autosomal trisomy syndrome in humans, but the detailed mechanism of its pathology remains unclear due to the lack of appropriate models of this disease. To resolve this problem, the current study reprogrammed human 18T amniotic fluid cells (AFCs) into an induced pluripotent stem cell (iPSC) line by introducing integration-free episomal vectors carrying pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. The pluripotency of 18T-iPSCs was subsequently validated by alkaline phosphatase staining, detection of iPSC biomarkers using real-time PCR and flow cytometry, detection of embryoid body (EB) formation, and detection of in vivo teratoma formation. Moreover, this study also investigated the transcriptomic profiles of 18T-iPSCs using RNA sequencing, and several gene clusters associated with the clinical manifestations of 18T were identified. In summary, the generated induced pluripotent stem cells line has typical pluripotency characteristics and can provide a useful tool with which to understand the development of 18T.

Establishment of cell lines for quality control of prenatal genetic diagnosis by SV40LT gene transfection

To establish a cell lines for quality control of prenatal genetic diagnosis. The recombined SV40LTag-pcDNA3.1(-) vector was constructed and transfected by lipidosome into human amniotic fluid cells with common aneuploidy. Positive clones were screened by G418, and the immortality of transfected cell line was identified. Cell line with karyotype of 46, XY, t(8;19)(q24.3;q13.1) from primary amniotic fluid cells was established. Karyotype analytical results indicated that the cell line at its 15th generation maintained the same karyotype of its primary cell. Gene SV40LT can lead to immortality of amniotic fluid cells, which contributes to preparing cell lines for internal and external quality control in prenatal genetic diagnosis.

The spontaneous differentiation and chromosome loss in iPSCs of human trisomy 18 syndrome

Aneuploidy including trisomy results in developmental disabilities and is the leading cause of miscarriages in humans. Unlike trisomy 21, pathogenic mechanisms of trisomy 18 remain unclear. Here, we successfully generated induced pluripotent stem cells (iPSCs) from human amniotic fluid cells (AFCs) with trisomy 18 pregnancies. We found that trisomy 18 iPSCs (18T-iPSCs) were prone to differentiate spontaneously. Intriguingly, 18T-iPSCs lost their extra 18 chromosomes and converted to diploid cells after 10 generations. fluorescence in situ hybridization analysis showed chromosome loss was a random event that might happen in any trisomic cells. Selection undifferentiated cells for passage accelerated the recovery of euploid cells. Overall, our findings indicate the genomic instability of trisomy 18 iPSCs bearing an extra chromosome 18.

ID:2041 Animal Component Free Medium Retains the Stability of Hypervariable Region 1 (HV1) of Mitochondrial DNA of Human Full Term Amniotic Fluid Mesenchymal Stem Cells

Therapeutic potential of human amniotic fluid (hAF) cells in treating human disease is very promising. Furthermore it provides an alternative in isolating stem cells from an accessible source, where the fluid is merely discarded, thus circumventing the ethical concerns. Mesenchymal stem cell (MSC) is one of the heterogenous cell populations in the amniotic fluid (AF), however, its role and potential for stem cell therapy are yet to be discovered. One of the criteria in establishing the clinical grade of MSCs is the ability of the cells to retain their genomic stability in vitro prior to bedside applications. The microenviroment niche of the culture medium used is important to generate the safe MSCs. As mitochondria play the vital role in metabolic task such as apoptosis and cell proliferation, therefore investigating mitochondria DNA (mtDNA) is essential to mark the stability of stem cell. In this study, we aimed to detect variations of mtDNA across the passage number of cultured human amniotic fluid MSCs (hAFMSCs), cultured in animal component free (ACF) medium, specialised for human MSCs. Polymorphisms of hypervariable region 1 (HV1) in the D-loop of mtDNA were investigated in a series of cultured hAFMSCs (passage 1 and 3) from three different samples (three biological replicates) as well as in their respective fresh samples (control). Extracted DNA samples were subjected to PCR to detect the sequence variations of HV1 (nt16024-16365). The sequences were then analysed using SeqScape v2.5 by comparing the sequences with The Cambridge Reference Sequence (rCRS) for human mtDNA. The common C-to-T transition at position 16223 (C16223T) for all three samples was identified. Another common base transition was also identified for the other two samples at position 16311 (T16311C).
 However, one of the samples showed more variants; a C-to-T transition at position 16290 (C16290T), an A-to-G at position 16317 (A16317G) and a G-to-A at position 16319 (G16319A). All variants were identified within the sequence of around 341bp nucleotides and their polymorphisms frequency was consistent for all three samples regardless of their passage numbers when compared to control. This pattern of polymorphisms demonstrates the HV1 mtDNA integrity of hAFMSCs cultured in ACF medium, hence highly suggesting the ability of ACF medium in retaining the stability of stem cells mtDNA in vitro. The ACF medium may serve as a good medium in culturing hAFMSCs that is safe and stable for basic research in stem cell and therapeutic applications.

Isolation and Molecular Characterization of Amniotic Fluid-Derived Mesenchymal Stem Cells Obtained from Caesarean Sections.

Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology, in vitro differentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFβ-signalling. An AF-MSC-enriched gene set included putative AF-MSC markers PSG5, EMX-2, and EVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling.

Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells

Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes

It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

First steps to define murine amniotic fluid stem cell microenvironment.

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells.

Effects of Pharmacological Agents on Human Amniotic Fluid-Derived Stem Cells in Culture.

Amniotic fluid contains multipotent cells and could be a source of stem cells for clinical use. Amniotic fluid cells (AFCs) are made up of a heterogeneous population of fetal cells that can be retrieved during pregnancy without ethical concerns as it is a standard clinical procedure. Studies of proliferation, multipotent marker expression, differentiation, and gene expression were performed after culturing with dexamethasone, valproic acid, and magnesium sulfate. There were correlations between known drug effects on the human fetus and changes seen in human AFCs in culture, as well as previously undescribed observations in neural and chondrogenic inducibility. Gene expression profiles confirmed these observations. AFC culture may provide a novel method to evaluate pharmacological agents before clinical use in pregnancy.

Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions.

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.