Human Somatic Stem Cells Research Articles

Expression profiles of human somatic mesenchymal stem cells derived from fresh endometrium, ectopic-endometrium and umbilical cord.

The study investigated the stem cell expression profiles and differentiation capacities of mesenchymal stem cells (MSCs) from different tissues, specifically human eutopic endometrium MSCs (eut-MSCs), ectopic endometrium MSCs (ect-MSCs), and umbilical cord MSCs (UC-MSCs). Our aim was to identify any similarities in subpopulations among these MSCs and lay a foundation for MSCs repair. MSCs were isolated from endometrial tissue (n = 5), endometriosis tissue (n = 6), and umbilical cords (n = 7). Flow cytometry was used to examine cell phenotype, and three lineage tests were conducted to evaluate the differentiation capacity of the MSCs. Eut-MSCs expressed CD44 (98.00 ± 0.96%), CD73 (99.54 ± 0.02%), CD140b (99.16 ± 0.50%), CD146 (93.87 ± 2.27%), SUSD2 (50.76 ± 8.15%), and CD271 (2.1 ± 1.22%). Ect-MSCs expressed CD44 (98.23 ± 1.60%), CD73 (99.63 ± 0.04%), CD140b (98.13 ± 0.53%), CD146 (93.88 ± 3.19%), SUSD2 (49.33 ± 6.36%), and CD271 (2.85 ± 1.17%). UC-MSCs expressed CD44 (99.11 ± ± 0.42%), CD73 (99.65 ± 0.12%), CD140b (99.84 ± 0.42%), CD146 (88.09 ± 4.20%), SUSD2 (72.87 ± 7.13%), and CD271 (6.19 ± 2.08%). The expression of SUSD2 and CD271 in UC-MSCs was slightly but not significantly higher than that in ect-MSCs and eut-MSCs. However, CD44, CD73, CD140b, and CD146 showed similar expression levels in UC-MSCs, ect-MSCs, and eut-MSCs. All three types of MSCs demonstrated the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. Our findings indicate that ect-MSCs, eut-MSCs, and UC-MSCs have similar stem cell phenotypes and the ability to differentiate into three lineages.

A systematic review on the effects of ROCK inhibitors on proliferation and/or differentiation in human somatic stem cells: A hypothesis that ROCK inhibitors support corneal endothelial healing via acting on the limbal stem cell niche.

Rho kinase inhibitors (ROCKi) have attracted growing multidisciplinary interest, particularly in Ophthalmology where the question as to how they promote corneal endothelial healing remains unresolved. Concurrently, stem cell biology has rapidly progressed in unravelling drivers of stem cell (SC) proliferation and differentiation, where mechanical niche factors and the actin cytoskeleton are increasingly recognized as key players. There is mounting evidence from the study of the peripheral corneal endothelium that supports the likelihood of an internal limbal stem cell niche. The possibility that ROCKi stimulate the endothelial SC niche has not been addressed. Furthermore, there is currently a paucity of data that directly evaluates whether ROCKi promotes corneal endothelial healing by acting on this limbal SC niche located near the transition zone. Therefore, we performed a systematic review examining the effects ROCKi on the proliferation and differentiation of human somatic SC, to provide insight into its effects on various human SC populations. An appraisal of electronic searches of four databases identified 1 in vivo and 58 in vitro studies (36 evaluated proliferation while 53 examined differentiation). Types of SC studied included mesenchymal (n=32), epithelial (n=11), epidermal (n=8), hematopoietic and other (n=8). The ROCK 1/2 selective inhibitor Y-27632 was used in almost all studies (n=58), while several studies evaluated ≥2 ROCKi (n=4) including fasudil, H-1152, and KD025. ROCKi significantly influenced human somatic SC proliferation in 81% of studies (29/36) and SC differentiation in 94% of studies (50/53). The present systemic review highlights that ROCKi are influential in regulating human SC proliferation and differentiation, and provides evidence to support the hypothesis that ROCKi promotes corneal endothelial division and maintenance via acting on the inner limbal SC niche.

In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture

Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O2 and CO2 with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that in vitro cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.

LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming.

Developing methods to improve the regenerative capacity of somatic stem cells (SSCs) is a major challenge in regenerative medicine. Here, we propose the forced expression of LIN28A as a method to modulate cellular metabolism, which in turn enhances self-renewal, differentiation capacities, and engraftment after transplantation of various human SSCs. Mechanistically, in undifferentiated/proliferating SSCs, LIN28A induced metabolic reprogramming from oxidative phosphorylation (OxPhos) to glycolysis by activating PDK1-mediated glycolysis-TCA/OxPhos uncoupling. Mitochondria were also reprogrammed into healthy/fused mitochondria with improved functional capacity. The reprogramming allows SSCs to undergo cell proliferation more extensively with low levels of oxidative and mitochondrial stress. When the PDK1-mediated uncoupling was untethered upon differentiation, LIN28A-SSCs differentiated more efficiently with an increase of OxPhos by utilizing the reprogrammed mitochondria. This study provides mechanistic and practical approaches of utilizing LIN28A and metabolic reprogramming in order to improve SSCs utility in regenerative medicine.

Rapid Production and Genetic Stability of Human Mesenchymal Progenitor Cells Derived from Human Somatic Cell Nuclear Transfer-Derived Pluripotent Stem Cells.

Pluripotent stem cell-derived mesenchymal progenitor cells (PSC-MPCs) are primarily derived through two main methods: three-dimensional (3D) embryoid body-platform (EB formation) and the 2D direct differentiation method. We recently established somatic cell nuclear transfer (SCNT)-PSC lines and showed their stemness. In the present study, we produced SCNT-PSC-MPCs using a novel direct differentiation method, and the characteristics, gene expression, and genetic stability of these MPCs were compared with those derived through EB formation. The recovery and purification of SCNT-PSC-Direct-MPCs were significantly accelerated compared to those of the SCNT-PSC-EB-MPCs, but both types of MPCs expressed typical surface markers and exhibited similar proliferation and differentiation potentials. Additionally, the analysis of gene expression patterns using microarrays showed very similar patterns. Moreover, array CGH analysis showed that both SCNT-PSC-Direct-MPCs and SCNT-PSC-EB-MPCs exhibited no significant differences in copy number variation (CNV) or single-nucleotide polymorphism (SNP) frequency. These results indicate that SCNT-PSC-Direct-MPCs exhibited high genetic stability even after rapid differentiation into MPCs, and the rate at which directly derived MPCs reached a sufficient number was higher than that of MPCs derived through the EB method. Therefore, we suggest that the direct method of differentiating MPCs from SCNT-PSCs can improve the efficacy of SCNT-PSCs applied to allogeneic transplantation.

In vitro evaluation of ferutinin on proliferation and osteogenesis differentiation in human unrestricted Somatic stem cells

Osteoporosis is a common disease in post-menopausal women. The increased risk of breast cancer and malignancy with hormone replacement, hampers its wide-usage. Phytoestrogens are known to have selective estrogen receptor modulator activity. The present study aims to determine how ferutinin affects unrestricted human Somatic Stem Cells (USSCs) osteogenic differentiation. The effect of ferutinin on USSCs proliferation was assessed by MTT assay while osteogenesis was evaluated using Alkaline Phosphatase Activity (ALP), calcium deposition and Alizarin Red Staining. Quantitative real-time PCR was applied to examine the expression of bone specific genes such as osteocalcin, Runx2, and BMP-2. Ferutinin (5–15 µg/mL) could positively impact on the proliferation of cells in a dose-dependent manner. Also, ALP enzyme activity and calcium deposition were enhanced in the presence of ferutinin. Based on real-time PCR results, ferutinin could increase the expression of bone marker genes. The pattern of ferutinin effect on gene expression is similar to standard synthetic estrogen, 17-β-estradiol. In the presence of the estrogen activity inhibitor (ICI), the effect of ferutinin on ALP and gene level was diminished. In conclusion, ferutinin may be considered as a potential candidate for the stem cell therapy in osteoporosis.

SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer

SIRT7, a sirtuin family member implicated in aging and disease, is a regulator of metabolism and stress responses. It remains elusive how human somatic stem cell populations might be impacted by SIRT7. Here, we found that SIRT7 expression declines during human mesenchymal stem cell (hMSC) aging and that SIRT7 deficiency accelerates senescence. Mechanistically, SIRT7 forms a complex with nuclear lamina proteins and heterochromatin proteins, thus maintaining the repressive state of heterochromatin at nuclear periphery. Accordingly, deficiency of SIRT7 results in loss of heterochromatin, de-repression of the LINE1 retrotransposon (LINE1), and activation of innate immune signaling via the cGAS-STING pathway. These aging-associated cellular defects were reversed by overexpression of heterochromatin proteins or treatment with a LINE1 targeted reverse-transcriptase inhibitor. Together, these findings highlight how SIRT7 safeguards chromatin architecture to control innate immune regulation and ensure geroprotection during stem cell aging.

Functional omics analyses reveal only minor effects of microRNAs on human somatic stem cell differentiation

The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up- and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNA-mediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC.

Human Cord Blood-Derived Unrestricted Somatic Stem Cell Infusion Improves Neurobehavioral Outcome in a Rabbit Model of Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth, which leads to hydrocephalus, cerebral palsy, and mental retardation. There are no available therapies to cure IVH, and standard treatment is supportive care. Unrestricted somatic stem cells (USSCs) from human cord blood have reparative effects in animal models of brain and spinal cord injuries. USSCs were administered to premature rabbit pups with IVH and their effects on white matter integrity and neurobehavioral performance were evaluated. USSCs were injected either via intracerebroventricular (ICV) or via intravenous (IV) routes in 3 days premature (term 32d) rabbit pups, 24 hours after glycerol‐induced IVH. The pups were sacrificed at postnatal days 3, 7, and 14 and effects were compared to glycerol‐treated but unaffected or nontreated control. Using in vivo live bioluminescence imaging and immunohistochemical analysis, injected cells were found in the injured parenchyma on day 3 when using the IV route compared to ICV where cells were found adjacent to the ventricle wall forming aggregates; we did not observe any adverse events from either route of administration. The injected USSCs were functionally associated with attenuated microglial infiltration, less apoptotic cell death, fewer reactive astrocytes, and diminished levels of key inflammatory cytokines (TNFα and IL1β). In addition, we observed better preservation of myelin fibers, increased myelin gene expression, and altered reactive astrocyte distribution in treated animals, and this was associated with improved locomotor function. Overall, our findings support the possibility that USSCs exert anti‐inflammatory effects in the injured brain mitigating many detrimental consequences associated with IVH. stem cells translational medicine 2019;8:1157–1169

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, bio-manufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.Lay abstractNovel therapies derived from different kinds of precursor cells and stem cells are increasingly moving to clinical trials to restore tissue function in patients who have suffered injury or disease. The manufacture of these new therapies is unusually complex, which means that the manufacturing processes require great attention to assure they are safe and effective. This paper describes a conversation amongst experts in the field who are exploring therapeutic applications of two different kinds of stem cells (pluripotent stem cells and tissue-derived stem/precursor cells). It considers critical issues in developing the manufacturing process for each of these quite different cells types.

Potentials of Differentiated Human Cord Blood-Derived Unrestricted Somatic Stem Cells in Treatment of Liver Cirrhosis.

Liver transplantation is the well-known treatment for chronic liver diseases; however, postoperative complications and lack of donors continue to be limitations with this treatment. Investigating new modalities for treatment of chronic liver illness is a must. In the present study, we aimed to clarify the effects of an in vitro hepatocyte-differentiated human unrestricted somatic stem cell transplant as a new cell-based therapy in an experimental model of chronic liver failure. Human umbilical cord blood-derived unrestricted somatic stem cells were isolated, cultured, propagated, and characterized. Cells were directed to differentiate into hepatocyte-like cells. An animal model of carbon tetrachloride cirrhotic liver failure was prepared, and the human in vitro differentiated unrestricted somatic stem cells were transplanted into the experimental model. Animals that did not receive transplant served as the pathologic control group. Animals were euthanized 12 weeks after transplant, and liver functions and histopathology were assessed. Compared with the pathologic control group, the transplant group showed improvements in levels of alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin. Histopathologic examination of the transplant group also showed improvements in hydropic degeneration and fibrosis. The use of unrestricted somatic stem cells, isolated and propagated from cord blood and then differentiated into hepatocyte-like cells, improved both fibrosis and normal function of cirrhotic livers. These cells could be considered as a line of cell-based therapy in cases of chronic liver disease.

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFβ signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFβ signaling was demonstrated in col7a1-/- mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)-9 and MMP-13. Furthermore, human cord blood-derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1-/- mice showed the ability to suppress TGFβ signaling and MMP-9 and MMP-13 expression meanwhile upregulating anti-fibrotic TGFβ3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient-derived fibroblasts, keratinocytes, and cSCC, respectively. The patient-derived cells were constitutively active for STAT, but not TGFβ signaling. Moreover, the levels of MMP-9 and MMP-13 were significantly elevated in the patient derived-keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP-9 and MMP-13, and interferon (IFN)-γ from RDEB patient-derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti-inflammatory and anti-fibrotic functions. Stem Cells 2018;36:1839-12.

Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.

Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.

Efficacy of Human Placental-Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal‐epidermal junction (DEJ). We recently demonstrated that human cord blood‐derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1 −/−) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental‐derived stem cells (HPDSCs), currently being used as human leukocyte antigen‐independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full‐term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1 −/− mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short‐term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1 −/− mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti‐C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. stem cells translational medicine 2018;7:530–542

Human Somatic Stem Cell Neural Differentiation Potential.

Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

Single-walled carbon nanotubes (SWCNTs) inhibit heat shock protein 90 (HSP90) signaling in human lung fibroblasts and keratinocytes

Single-walled carbon nanotubes (SWCNTs) are carbon-based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1–3μm) and regular-length (5–30μm) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type-dependent manner. The toxicity of regular-length pristine SWCNT was most evidenced in NP69>CYT00086>MCF-10A>MRC-5>HaCaT > HEK-293T>HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular-length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular-length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC-5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC-5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC-5 cells to SWCNTs results in a dose-dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90-dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT-induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes.

Application of cell growth analysis to the quality assessment of human cell-processed therapeutic products as a testing method for immortalized cellular impurities.

In human cell-processed therapeutic products (hCTPs) for clinical application, tumorigenic cellular impurities in the manufacturing process are a major concern. Because cellular immortalization is one of the prerequisite steps in tumorigenesis, we tested whether cell growth analysis can be employed to check for immortalized (and potentially tumorigenic) cellular impurities in hCTPs. We monitored the growth of human bone marrow-derived mesenchymal stem cells (BMSCs) mixed with HeLa cells at a ratio of 1/106 or more and compared their growth rates with that of BMSCs alone. The cell growth analysis detected a significant increase in the growth rate of the BMSCs spiked with 0.0001% HeLa within 30 days at a probability of 47%. When human adipose-derived stem cells (ADSCs) were spiked with ASC52telo cells, a human telomerase reverse transcriptase (hTERT)-immortalized adipose-derived mesenchymal stem cell line, at a ratio of 0.001% or more, their growth rates were significantly increased within 15 passages, compared with that of ADSCs alone. These results indicate that cell growth analysis for the detection of immortalized cellular impurities in human somatic stem cells is simple and can be useful for the quality assessment of hCTPs in the manufacturing process.

Structural and quantitative evidence of \u03b12\u20136-sialylated N-glycans as markers of the differentiation potential of human mesenchymal stem cells

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such, they hold promise for use in stem cell-based therapies. However, no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously, we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that α2–6-sialylation is a marker of the differentiation potential of these cells. Nevertheless, no information was available about the structural details of these of α2–6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs), human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of α2–6-sialylated N-glycans was detected in early passage cells (24–28 % of sialylated N-glycans) compared with late passage cells (13–15 %). A major α2–6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes, Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast, no significant differences were observed between early and late passage hMSCs with respect to α2–6-sialylated O-glycan percentages. These results demonstrate that levels of α2–6-sialylated N-glycans, but not O-glycans, could be used as markers of the differential potential of hMSCs.

Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203.

Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) treated with a retina differentiation cocktail induced gene expressions of retina development-relevant genes. Furthermore, microRNA-203 (miR-203) is abundantly expressed in human AESCs and human UCB-MSCs. This miR-203 is predicted to target multiple retina development-relevant genes, particularly DKK1, CRX, RORβ, NEUROD1, NRL and THRB. The inhibition of miR-203 induced a retina differentiation of AESCs and UCB-MSCs. Moreover, successive treatments of anti-miR-203 led to the expression of both mature photoreceptor (PR) markers, rhodopsin and opsin. In addition, we determined that CRX, NRL and DKK1 are direct targets of miR-203 using a luciferase assay. Thus, the work presented here suggests that somatic stem cells can potentially differentiate into neural retina cell types when treated with anti-miR-203. They may prove to be a source of both PR subtypes for future allogeneic stem cell-based therapies of non-regenerative retina diseases.