T. A. Krylova

Comparative characteristics of new lines of mesenchymal stem cells derived from human embryonic stem cells, bone marrow, and foreskin

New human nonimmortalized fibroblast-like cell lines were derived from various sources: from embryonic stem cells (ESCs) (the SC5-MSC and SC3a-MSC lines), from bone marrow of a 5- to 6-week-old fetus (the FetMSC line), and from foreskin of a 3-year-old child (the FRSN line). All the lines are successfully used as feeders during cultivation of human ESCs. The mean doubling time of the cell populations fluctuates depending on the line from 25.5 h in the SC5-MSC line to 38.8 h in the SC3a-MSC line. The growth curves indicate active cell proliferation of all lines. Numerical and structural karyotypical analysis has shown these lines to have a normal karyotype: 46, XX (SC5-MSC and SC3a-MSC) and 46, XY (FetMSC and FRSN). To determine the status of these lines, comparative analysis of surface markers was performed with the aid of flow cytofluorimetry, and expression of the c antigens characteristic of human mesenchymal stem cells (MSCs) was revealed: CD44, CD73, CD90, CD105, and HLA-ABC and the absence of expression of CD34 and HLA-DR. Interlinear differences in the expression level of the marker CD117 (c-kit) were revealed. Immunofluorescent and cytofluorimetric analysis of expression of surface markers and transcription factor Oct-4 that are characteristic of human ESCs has shown that, in all four lines, expression of TRA-1-60 and Oct-4 is absent, whereas in expression of SSEA-4 there are observed the interlinear differences not depending on the origin of cells. At present it is not yet clear whether the revealed interlinear differences affect essentially the functional status of mesenchymal stem cells. Immunofluorescent analysis in cells of all lines showed expression of markers of early differentiation into derivatives of three germ layers characterizing ESCs, which might possibly provide wide MSC possibilities during reparation of different tissue damages, depending on the corresponding microenvironment. The capability of cells of all lines for directed differentiation into the adipogenic and osteogenic directions was revealed.

A comparative analysis of mesenchymal stem-cell lines derived from bone marrow and limb muscle of early human embryos

The properties of mesenchymal stem-cell (MSC) lines derived from bone marrow (FetMSC) and limb muscle (M-FetMSC) of 5- to 6-week human embryos were assayed. The main cell-line characteristics were obtained at the sixth passage. The average cell-population doubling time was 33.0 ± 1.4 h for FetMSC and 25.0 ± 0.1 h for M-FetMSC. Growth curves indicated active cell proliferation. Numerical and structural karyotypic analysis showed that these lines have normal karyotype, 46, XY. Cell surface markers were analyzed by flow cytometry. It was found that the cells express CD44, CD73, CD90, CD105, and HLA-ABC surface antigens and vimentin, which are common for human MSC beings, and lack CD34 and HLA-DR. The cells were capable of osteogenic, chondrogenic, and adipogenic differentiation. Immunofluorescence and flow-cytometry assays reveled a lack of surface antigen TRA-1-60, high expression of surface antigen SSEA-4, and low expression of transcription factor Oct-4 attributed to human embryonic stem cells. Immunofluorescence analysis showed the presence of early differentiation markers, three germ-layer derivatives for human embryonic stem cells. This makes MSCs useful for repair of damaged tissue in the corresponding microenvironment. Despite their shared MSC status, the FetMSC and M-FetMSC lines displayed some interlinear differences related to growth characteristics and differentiation potential. The MFetMSC line exhibited reduced potential for adipogenic differentiation compared to the FetMSC line. Immunofluorescence analysis revealed Z-disks in M-FetMSC, but not in FetMSC, during skeletal-muscle differentiation. These findings suggest that the different microenvironment has an influence when cells are in an organism before their transplantation in vitro.

[Comparative characteristics of new human embryonic stem cell lines SC5, SC6, SC7, and SC3a].

Numerous human embryonic stem cell lines with different genetic background are widely used as cell models for fundamental, biomedical, and pharmacological research. New hES cell lines SC5, SC6, SC7, and SC3a are derived from the blastocysts and maintained on mitotically inactivated human feeder cells. All derived hES cell lines passed through more than 120 cell population doublings, retained normal diploid karyotype and ability for in vitro differentiation in the derivates of three germ layers. These lines express the markers of undifferentiated hES cells: Oct-4, Nanog, SSEA-4, TRA-1-60, and alkaline phosphatase. Moreover, undifferentiated cells of SC5, SC6, and SC7 lines expressed germ line specific genes DPPA3/STELLA and DAZL and did not express somatic lineage specific genes. In contrast, undifferentiated cells of the SC3a line did not express DPPA3/STELLA and DAZL but expressed extra embryonic endoderm cell markers GATA4 and AFP. Double staining of SC5 and SC3a colonies by antibodies against transcription factors Oct-4 and GATA4 has demonstrated that most SC3a cells in colonies were positive for both factors. Furthermore, the cells of SC5, SC6, and SC7 lines but not of the SC3a line formed teratomas containing derivates of the three germ layers. These results indicate that, in contrast to the other cell lines, the cells in the SC3a colonies represent an early committed cell population. Moreover, expression of the multidrug resistance transporter gene ABCG2 was detected in undifferentiated cells and differentiated embryonic bodies (EB) of all lines during 10 days by immunofluorescent and RT-PCR analysis, whereas RT-PCR analysis has revealed up-regulation of the ABCB1 transporter gene expression in differentiating embryoid bodies of SC5, SC6, and SC7 cells only. Thus, these findings demonstrate different characteristics and differentiation potential of SC5, SC6, SC7, and SC3a hES cell lines, which were derived in different conditions.

Cellular spheroids obtained from mesenchymal stem cells derived from bone marrow and limb muscle of early human embryo

Cellular spheroids were obtained from mesenchymal stem cells derived from different tissues of 5to 6-week-old embryos: bone marrow (FetMSC) and limb muscle (M-FetMSC). The properties of these cells maintained in monolayer (2D cultivation) or spheroids (3D cultivation) were compared. Cellular spheroids were obtained from monolayer cultures at the sixth passage after decryopreservation and assayed 48 h after their formation. Unlike monolayer cultures, spheroids are heterogeneous cell populations consisted of fibroblast-like and epithelioid cells. Two-day-old spheroids are actively proliferating structures. Cell surface markers were analyzed by flow cytometry. Monolayer culture and cellular spheroids expressed CD44, CD73, CD90, CD105, and HLA-ABC common for mesenchymal stem cells (MSCs) and lacked CD34 and HLA-DR. However, expression of CD90 and CD105 antigens was significantly lower in spheroids than in monolayer cultures. Expression of transcription factors and surface antigens typical for human embryonic stem cells were analyzed with immunofluorescence and flow cytometry. Expression of Sox-2 and SSEA-4 in 2D and 3D cultures, lack of Oct-4 in 2D cultures, and its increase in 3D cultures were found. Both monolayer cells and spheroids were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages, although monolayer cultures and spheroids differed. Adipogenic differentiation was more active in the cellular spheroids from M-FetMSC cells compared to corresponding monolayer cultures. Differences between the 2D and 3D cultures of both lines have been shown by the character of chondrogenic differentiation. As a whole, these findings confirm the MSC status for the cellular spheroids derived from FetMSC and M-FetMSC monolayer cultures and attest to their increased differentiation potential compared to monolayer cultures.

The Dynamics of Cell Properties during Long-Term Cultivation of Two Lines of Mesenchymal Stem Cells Derived from Wharton’s Jelly of Human Umbilical Cord

The cell properties of two MSC lines (MSCWJ-1 and MSCWJ-2) established from Wharton’s jelly of human umbilical cord were analyzed at the 6th, 13th, and 28th passages. The following were discovered. (1) The cell morphology during long-term cultivation was changed in both lines, with the cells being larger and having more debris in the MSCWJ-2 line than in the MSCWJ-1 line. (2) As shown by analysis of growth characteristics, long-term cultivation was accompanied with a significant increase of the average population-doubling time in both cell lines. This was correlated with morphological changes and a gradual increase in β-galactosidase activity, indicating the occurrence of replicative senescence. The interline changes concerned the level of proliferative activity, the index of proliferation, the time of the entry into the phase of replicative senescence (30 population doublings in the MSCWJ-2 line and 60 population doublings in the MSCWJ-1 line), and the percentage of senescent cells (80% in MSCWJ-2 line compared to 58% in the MSCWJ-1 line). (3) Numerical karyotypic analysis showed that both cell lines at early and late passages had normal karyotype 46, XX (MSCWJ-1 cells), and 46, XY (MSCWJ-2 cells). Structural karyotypic analysis showed a cytogenetic heterogeneity in both lines: low frequency of random clonal and nonclonal chromosomal rearrangements disappeared during long-term cultivation. Unlike in the case of the MSCWJ-1 line, the clonal chromosomal rearrangement of the short arm of chromosome 7 with a frequency of 39% was detected at the sixth passage in the MSCWJ-2 line but disappeared at the 13th passage. (4) Surface marker analysis of these cell lines at the 6th, 13th, and 28th passages revealed that these cells expressed surface antigens characteristic of human MSC (CD44, CD73, CD90, CD105, HLA-ABC), as well as vimentin, and did not express CD34 and HLA-DR. (5) Analysis of nondifferentiated hESC markers in both lines showed a decrease in the level of expression of the surface marker SSEA-4 and a lack of expression of the transcription factor SOX-2 at the 13th passage in the MSCWJ-2 and at the 28th passage in MSCWJ-1 cells. (6) Cell adhesion in both lines was lower in late passages compared to the cells at the sixth passage. The cells retained an osteogenic and adipogenic differentiation potential at late passages. A lack of chondrogenic differentiation in MSCWJ-2 cells, but not in MSCWJ-1 cells, was registered at late passages. The MSCWJ-2 cell line exhibited a frequently occurring clonal chromosomal rearrangement at an early passage, early replicative senescence with a large proportion of senescent cells, a significantly decreased index of proliferation during long-term cultivation, a decrease or disappearance of the expression of nondifferentiated ESCs of markers, and decreased differentiation potential. Given the metabolic cooperation between the cells, we can cautiously suppose that the clonal chromosomal rearrangement of the short arm of chromosome 7 detected at an early passage with high frequency is coupled with activation of cellular processes that proceed in the absence of this chromosomal aberration.

Characteristics and specific features of new human embryonic stem cell lines

Four continuous human embryonic stem cell (ESC) lines (SC1, SC2, SC3, and SC4) isolated from preimplantational blastocysts obtained by artificial fertilization have been described. The cell lines were cultivated on mitotically inactivated human feeder cells. SC1 and SC2 cell lines passed through 150 population doublings, while the SC3 and SC4 cell lines passed through about 120 population doublings, which significantly exceeded Hayflick’s limit. All of these cell lines maintain the high activity of alkaline phosphatase and the expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60, TRA-1-81), which confirms the status of ESC and human specificity. An immunofluorescent analysis of the expression of antigens that characterize the ectoderm, endoderm, and mesoderm has confirmed the capacity of these cells for pluripotency in all four lines under in vitro conditions. Using PCR analysis, the expression of six genes specific to pluripotent cells, i.e., OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO, and LEFTYA, has been revealed. The correlation between the level of proliferative activity and the character of staining with DNA-bound fluorescent dyes has been found. Hoechst 33342 and PI dyes produced a diffuse staining of nuclei in slowly proliferating cells of the SC1 and SC2 lines. On the contrary, in actively proliferating cells of the SC3 and SC4 lines, a distinct staining of nuclei was observed. Upon changing the cultivation conditions, proliferative activity of the SC3 and SC4 lines decreased and the character of the fluorescent staining became similar to that of the SC1 and SC2 lines. The quality of the fluorescent staining with Hoechst 33342 and propidium iodide reflects the level of proliferation. Possible causes and mechanisms of this peculiarity of human ESCs are discussed.

Properties of stem cells isolated from subcutaneous and subepicardial adipose tissues

Stem cells (SCs) vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. Comparative analysis of their biological properties is essential for making an optimal SC choice for regenerative therapy. Using immunocytochemistry, flow cytometry, histochemistry, and RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissues and cultured under similar conditions without any differentiation-promoting factors. The cultures were similar in having a high proportion of proliferating cells positive for nuclear antigen (PCNA). In both cultures, immunophenotyping has revealed high expression of mesenchymal stem-cell surface markers CD29, CD44, CD73, and CD105; low expression of CD31, CD34, and CD45; and variability in CD117, CD146, and CD309 expression. The only difference in the CD marker profile was the significantly lower expression of CD90 in the culture of SCs from SC-AT than from SEC-AT. Histochemical analysis showed a lack of Oil Red O-positive cells in both cultures and an about ten times higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In both cultures, immunocytochemistry detected low expression of the slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Expression of the gap junction protein connexin-43 was markedly higher in cells from SC-AT cultures. Only the cells of these cultures expressed the epithelial cell marker cytokeratin-19. GATA4 mRNA expression detected with RT-PCR was identified in SEC-AT rather than in SC-AT cells. Our results suggest that SC-AT is enriched compared to SEC-AT with epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs and can be considered an alternative source for cell cardiotherapy.

The effect of type I collagen and fibronectin on the morphology of human mesenchymal stromal cells in culture

Limited knowledge of the behavior of stem cells in culture seems to be one of the reasons for problems in their successful introduction to applied medicine. To address this issue, we studied in vitro interaction of human mesenchymal stromal cells (MSCs) with various substrates (plastic, type I collagen, fibronectin, and mixtures of these proteins at various ratios) for 16–18 h after cell plating. Several cell-morphology features, such as area, perimeter, spreading coefficient, and polarization coefficient were determined. It is shown that MSCs specifically respond to the substrate and consist of several groups. Collagen and fibronectin have opposite effects on polarization and spreading of the cells. Collagen preferably enhances polarization of the cells, whereas fibronectin stimulates proportional spreading of cells. The combined effect on the cells of the collagen-fibronectin mixture cannot be considered as a simple additive effect. We assume that variations in the ratio of ECM proteins may be one mechanism of a directed action on stem cells.

Two spreading states of human embryo mesenchymal cells in vitro

Spreading of mesenchymal cells of human embryo on plastic and on type I collagens (from rat, sheep, and ox) was studied. Spreading of the cells on collagens was stronger than that in control, but no differences between different collagens were revealed. The cell perimeter, the spreading coefficient, and the cell projection area on the substrate were used as morphometric parameters. The spreading of cells was monitored for 0.5–2 h after plating. During the spreading both on plastic and on collagen, groups of small cells were revealed as separate subpopulations. As a whole, such cells accounted for 9% of the cell population in control and for 2% in experiment. We assume that this cell type is associated with a special independent functional state of the cells that precedes cell spreading.

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.