A novel feeder-free system for human embryonic stem cells and characterization of their sublines with autogenic and allogenic cultivation

We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.     

Establishment and characteristics of new human embryonic stem cell subline SC6-FF in an allogenic feeder-free culture system

The novel human embryonic stem cell (hESC) subline SC6-FF was derived from SC6 cells in an allogenic feeder-free culture system. Key components of the feeder-free culture system were extracellular matrix proteins and conditioned medium from the mesenchymal stem cell line SC5-MSC. These conditions are allogenic for SC6-FF cells. SC6-FF subline underwent more than one hundred cell population doublings and retained a normal diploid karyotype; 46, XX. The average population doubling time was 23.7 ± 0.8 h, similar to that of the parent SC6 line. The presence of undifferentiated hESC markers (alkaline phosphatase activity, Oct-4, SSEA-4, and TRA-1-60) was verified by histochemistry and immunofluorescence. Cells were distinguished from parental cells in size and morphology as a result of spontaneous differentiation. These cells exhibited the ability to differentiate into derivates of three germ layers by expressing common markers of the ectoderm (alpha-fetoprotein), mesoderm (a-actinin) and endoderm (a-fetoprotein) cells. We could conclude that characteristics of the novel feeder-free SC6-FF subline correspond to the status of human embryonic stem cells.     

Human embryonic stem cells: Problems and perspectives

Generation of human embryonic stem cell lines is one of the most important achievements in biological science in the 20th century. It has excited a wide scientific and social response, as embryonic stem cells (ESC) may, in the future, be regarded as an unlimited source of transplantation materials for replacement cell therapy. ESC lines are derived, cultured, inner cell mass from human blastocysts is used in the in vitro fertilization procedure. To date, human embryonic cell lines have been obtained in more than 20 countries. In our country, embryonic stem cell research is carried out in the Institute of Cytology, Russian Academy of Sciences and the Institute of Gene Biology, Russian Academy of Sciences. Studies with human ESC go in several directions. Much attention is paid to finding the most optimal conditions for ESC cultivation, mainly to the development of cultivation techniques excluding animal feeder cells and other components of animal origin. Another direction is a large-scale analysis of gene expression specific to the embryonic state of cells and the corresponding signaling pathways. Great efforts are being focused on the directed differentiation of ESC into various tissue-specific cells. It has been shown that in vitro ESC are able to differentiate into virtually any somatic cells. Works are in progress to develop methods for “therapeutic cloning,” i.e. the transfer of somatic nuclei into enucleated oocytes or embryonic stem cell cytoblasts and their reactivation. Of great importance is the standardization of the human ESC lines. However, standard requirements for cells utilized for research or therapeutic purposes may be different. It has been found that many permanent human ESC lines underwent genetic and epigenetic variations. Therefore, the cell line genetic stability should be periodically verified. The main purpose of the review is to provide a detailed consideration of research on the genetic stability of human and mouse ESC lines. Human ESC lines established both in our country and others could not thus far be used in clinical practice. It is highly probable that undifferentiated ESCs cannot be applied for therapeutic purposes, as there is a risk of their malignant transformation. Therefore, main efforts should be focused on the production ESC progenitor and highly differentiated cells suitable for transplantation.     

Multipotent mesenchymal stem cells of desquamated endometrium: isolation, characterization and use as feeder layer for maintenance of human embryonic stem cell lines

In this study, we characterize new multipotent human mesenchymal stem cell (MSC) lines derived from desquamated (shedding) endometrium in menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSC of any origin. The eMSCs have positive expression of CD73, CD90, CD105, CD13, CD29, CD44 markers and the absence of expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130 and HLA-DR (class II). Multipotency of the established eMSC is confirmed by their ability to differentiate into other mesodermal cell types such as osteocytes and adipocytes. Besides, the isolated eMSC lines partially (over 50%) express the pluripotency marker SSEA-4, but do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and beta-III-tubulin. This suggests a neural predisposition of the established eMSC. These cells are characterized by high rate of cell proliferation (doubling time 22-23 h) and high cloning efficiency (about 60%). In vitro the eMSCs undergo more than 45 population doublings revealing normal karyotype without karyotipic abnormalilies. We demonstrate, that the mititotically inactivated eMSCs are perfect feeder cells for human embryonic stem cell lines (hESC) C612 and C910. The eMSC being a feeder culture maintain the pluripotent status of the hESC, which is revealed by the expression of Oct-4, alkaline phosphatase and SSEA-4. When co-culturing, hESC retain their morphology, proliferative rate for more than 40 passages and capability for spontaneous differentiation into embryoid bodies comprising the three embryonic germ layers. Thus, an easy and non-invasive extraction of the eMSC in menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESC to clinical setting.     

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.     

Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.     

Human embryonic stem cells. Problems and perspectives

Establishment of human embryonic stem cell lines is one the major achievements in the biological science in the XX century and has excited a wide scientific and social response as embryonic stem cells can be regarded in future as unlimited source of transplantation materials for the replacement cell therapy. To date human embryonic cell lines are obtained in more than 20 countries. In our country the embryonic stem cell researches are carried out in the Institute of Cytology RAS and the Institute of Gene Biology RAS. ESC lines are derived from placed in culture inner cell mass of human preimplantation blastocysts used in the in vitro fertilization procedure. Studies with human ESC go in several directions. Much attention is paid to the elaboration of the optimal conditions for ESC cultivation, mainly to the development of cultivation methods excluding animal feeder cells and other components of animal origin. Another direction is a scale analysis of gene expression specific for the embryonic state of the cells and corresponding signaling pathways. Many efforts are concentrated to find conditions for the directed differentiation of ESC into different tissue-specific cells. It has been shown that ESC are able to differentiate in vitro practically into any somatic cells. Some works are initiated to develop methods for the "therapeutic cloning", that is transfer and reactivation of somatic nuclei into enucleated oocytes or embryonic stem cell cytoblasts. Of great importance is human ESC line standardization. However, the standard requirements for the cells projected for research or therapeutic purposes may be different. It has been found that many permanent human ESC lines undergo genetic and epigenetic changes and, therefore, the cell line genetic stability should be periodically verified. The main aim of the review presented is a detailed consideration of the works analyzing the genetic stability of human and mouse ESC lines. Human ESC lines established in our and as well as in other countries couldn't be used so far in clinical practice. It is highly probable that undifferentiated ESC cannot be applied for therapeutic purposes because of the risk of their malignant transformation. Therefore, main efforts should be focused on the production of progenitor and highly differentiated cells suitable for transplantation derived from ESC.     

Isolation and Comparative Characteristics of Mesenchymal Stem-Cell Lines Derived from Foreskin of Two Donors of Similar Age

Two new nonimmortalized human cell lines FRSN-1 and FRSN-2 were established from foreskin of two similarly aged donors (2.5 years). Growth characteristics and differentiation potential of these cell lines studied on the sixth passage confirmed their status as mesenchymal stem cells (MSCs). A number of characteristics have been analyzed during long-term cultivation up to the 26th passage. The dynamics of the process of replicative senescence defined by the activity of β-galactosidase differed between these lines. However, at the 26th passage, the process of replicative senescence was equally enhanced in both lines. The plating efficiency markedly differed between the lines on the sixth passage. In FRSN-1, it was higher than in FRSN-2. The plating efficiency substantially dropped to the 26th passage in FRSN-1 and was lost in FRSN-2 line. Growth curves showed active proliferation of these lines at the 6th passage. The average doubling time did not differ between the lines and was 36.9 and 39.0 h, respectively. Analysis of growth curves on the 26th passage revealed a decline in proliferative activity and increase in average doubling time of cell populations in both lines, more in FRSN-2 than in FRSN-1 lines. The patterns of growth curves differed in these lines. Morphological analysis revealed increased cell size and spreading typical for the phase of the replicative senescence. Numerical and structural karyotypic analysis at the sixth passage showed that both lines have normal karyotype 46, XY. We did not discover interline differences in the frequency of chromosomal aberrations. To determine the status of these cell lines, comparative analysis of the surface markers was performed using flow cytometry. It was revealed that cells of both lines expressed surface antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, and HLA-ABC and did not express CD34, CD45 and HLADR. Cells of both lines displayed SSEA-4 and SOX2, markers of human embryonic stem cells (ESCs). Expression of SSEA-4 was also detected at the 26th passage in both lines. FRSN-1 and FRSN-2 cells expressed the markers of early ESC differentiation into three germ layers. The ability of these cell lines to differentiate into osteogenic, chondrogenic, and adipogenic lineages was shown on the sixth passage. Both lines exhibited substantially reduced adipogenic potential on the 20th passage. These data indicate that in contrast to growth characteristics the adipogenic differentiation potential changes even with an average degree of replicative senescence. It appears that the cell replicative senescence contributed to the change in MSC differentiation potential. Overall, the results demonstrate that cell lines derived from different donors are distinguished in growth characteristics and pattern of replicative senescence. The disparity is due to a direct genetic influence and indirectly by different microenvironment in their donor organisms before cell isolation.     

Novel human embryonic stem cell lines C612 and C910

Novel human embryonic stem cell lines C612 and C910 have been established from atching blastocytes. Cells were cultivated in mTeSR medium on a mouse fibroblast feeder layer; they exhibit common pluripotent markers, such as alkaline phosphatase, Oct 3/4, SSEA-4, Nanog, Rex1. The immunophenotyping of these cells by flow cytometry revealed CD90 (Thy-1) and CD117 (c-kit) antigens and showed weak or no expression of CD13, CD34, CD45, CD130, and HLA class I and II antigens, which is typical for human embryonic stem cells. Karyotypic structure of C612 and C910 assayed by the G-banding of metaphase plates is normal in both chromosome number and structure. The cells generate embryoid bodies, undergo spontaneous differentiation, and express three germ-layer markers (nestin, keratin, vimentin ectoderm), α-fetoprotein (entoderm), muscle α-actinin (mesoderm), i.e., possess pluripotent potency. Thus, C612 and C910 display accepted human embryonic stem cell properties, including unlimited self-renewal, expression of pluripotent markers, ability to differentiate into three germ layers, and are diploid; therefore, they may be of potential use for fundamental research, as well as for replacement therapy studies.     

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.     

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.     

Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct-4 and Rex-1

BACKGROUND: Previous adult stem cells studies have provided evidence that BM mesenchymal stem cells (MSC) exhibit multilineage differentiation capacity. These properties of MSC prompted us to explore the neural potential of MSC with a view to their use for the treatment of demyelinating disorders, such as multiple sclerosis. Indeed, issues such as the identification of a subset of stem cells that is neurally fated, methods of expansion and optimal stage of differentiation for transplantation remain poorly understood. METHODS: In order to isolate mouse (m) MSC from BM, we used and compared the classic plastic-adhesion method and one depleting technique, the magnetic-activated cell sorting technique. RESULTS: We established and optimized culture conditions so that mMSC could be expanded for more than 360 days and 50 passages. We also demonstrated that undifferentiated mMSC express the neural markers nestin, MAP2, A2B5, GFAP, MBP, CNPase, GalC, O1 under standard culture conditions before transplantation. The pluripotent stem cell marker Oct-4 and the embryonic stem cell marker Rex-1 are spontaneously expressed by untreated mMSC. The lineage-negative mMSC (CD5- CD11b- Ly-6G- Ter119- CD45R- c-kit/CD117-) overexpressed Oct-4, O1 and A2B5 in the first days of culture compared with the non-sorted MSC. Finally, we identified a distinct subpopulation of mMSC that is primed towards a neural fate, namely Sca-1+/nestin+ mMSC. DISCUSSION: These results should facilitate the optimal timing of harvesting a neurally fated subpopulation of mMSC for transplantation into animal models of human brain diseases.