Jerzy Kawiak

Restricted Myogenic Potential of Mesenchymal Stromal Cells Isolated From Umbilical Cord

Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Whartons jelly have been shown to be able to differentiate into various cell types. Thus, as they are readily available and do not raise any ethical issues, these cells are considered to be a potential source of material that can be used in regenerative medicine. In our previous study, we tested the potential of whole mononucleated fraction of human umbilical cord blood cells and showed that they are able to participate in the regeneration of injured mouse skeletal muscle. In the current study, we focused at the umbilical cord mesenchymal stromal cells isolated from Whartons jelly. We documented that limited fraction of these cells express markers of pluripotent and myogenic cells. Moreover, they are able to undergo myogenic differentiation in vitro, as proved by coculture with C2C12 myoblasts. They also colonize injured skeletal muscle and, with low frequency, participate in the formation of new muscle fibers. Pretreatment of Whartons jelly mesenchymal stromal cells with SDF-1 has no impact on their incorporation into regenerating muscle fibers but significantly increased muscle mass. As a result, transplantation of mesenchymal stromal cells enhances the skeletal muscle regeneration.

ENCAPSULATION OF OKT3 CELLS IN HOLLOW FIBERS

Encapsulation of an OKT3 cell line in hollow fibers was evaluated in vitro and in vivo. The cell line is a mouse hybridoma producing immunoglobulin G2a (IgG2a) against CD3 human T lymphocytes and thus may function as a nonspecific activation system of a subpopulation of human T lymphocytes. For encapsulation purpose, hollow fibers of polypropylene K600 PP Accurel (Akzo, Germany) were selected. Hollow fibers were siliconized to improve membrane biocompatibility for in vivo experiments. The siliconized hollow fibers exhibited acceptable diffusive permeability (P) [ml/min/m2] for small solutes (for creatinine, p = 63.9 +/- 2.0, n = 3) and larger solutes (for albumin, p = 16.9 +/- 1.9, n = 3; for IgG, p = 1.0 +/- 0.2, n = 3). The 12 cm long hollow fibers were filled with a suspension of OKT3 cells of an average density of 10(6) cells/ ml, and both ends were sealed. The encapsulated cells were cultivated in RPMI 1640/10% CS medium at 37 degrees C, 5% CO2 for a period of 3 to 4 days. After the culture period, the medium was tested on human peripheral blood lymphocytes for the presence of anti-CD3 antibody and read in a flow FACS-trac cytometer (Becton Dickinson Immunocytochemistry Systems, San Diego, CA). The tightness of hollow fiber sealing was tested with a bubble point method. The number of cells increased after cultivation by four- to nine-fold on average (n = 11). Ten experiments were performed in vivo with OKT3 cells encapsulated in hollow fibers and implanted subcutaneously into mice for 3 days. In 50% of the experiments, some anti-CD3 antigens on human lymphocytes were found; however, the difference, in comparison with control, in percent of CD3+ was insignificant. In conclusion, the hollow fiber method for cultivation of hybridoma cells in vitro allows for separation of cells from the medium containing secreted anti-CD3 antibodies and is effective in maintaining cell viability. In vivo application needs additional study.

Polypropylene hollow fiber for cells isolation: methods for evaluation of diffusive transport and quality of cells encapsulation.

Formulation of membrane properties is important prior the successful implantation of encapsulated cells producing therapeutically relevant compounds. The purpose of our study was to specify the methods allowing preliminary evaluation of hollow fibers (HF) chosen for immunoisolation. We have selected as estimates (1) diffusive permeability for small and large solutes, and HF cut off (in vitro), (2) histological evaluation of tissue overgrowth after sc. implantation into mice. It was found that diffusive coefficients were linearly dependent on the particle diameter except that of albumin (2–3 times higher than theoretically estimated). This discrepancy imply that for certain particles the interaction with membrane material may be significant. The histological evaluation showed that siliconized HF implanted for 105 days were accepted (there was thin fibrotic layer on the external surface of the HF, no surrounding haemopoietic cells were found). It is concluded that proposed methods for preliminary evaluation of hollow fibers chosen for immunoisolation seems to be reliable and suitable for testing diffusive permeability of each relevant cell product.

Growth factors and their receptors derived from human amniotic cells in vitro.

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine.

Monitoring cell proliferation in vitro with different cellular fluorescent dyes.

There are few methods for quantifying cell proliferation. Those tests describe the proliferation kinetics of a cell population, but they do not report the history of single cells, the number and frequency of cell divisions, or the precursor cell frequency. Cell-tracking assays based on dilution of the green fluorescent protein labelling dye, CFSE, has become the standard for monitoring cell proliferation. Other labelling dyes, e.g. CellTrace Violet and CellVue Claret, are also used for the same purpose. This study aimed to compare these three cell labelling methods for analysing the kinetics of cell viability, proliferation, and precursor cell frequency. Human peripheral blood mononuclear cells stimulated with Concanavalin A (ConA) were used as a model system. After labelling with a cell-tracking dye cells were divided into groups with and without ConA stimulation. From the 5th to 8th day, cells were collected and analysed with flow cytometry. Cell viability was not significantly different between labelled and unlabelled cells that received ConA stimulation. The proliferative fraction, proliferation index, and nonproliferative fraction were not significantly different among lymphocytes labelled with different dyes. Precursor cell frequency was also similar among cells labelled with the three cell-tracing dyes. The practical conclusion from our observations is that the results from cells labelled with different tracers may be compared directly and discussed jointly.

Granulocyte-macrophage colony-stimulating factor potentiates antitumor activity of interleukin-12 in melanoma model in mice.

To study the antitumor activity of the combination immunotherapy with interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF), a murine MmB16 melanoma tumor model was used. Seven days after inoculation of MmB16 melanoma cells into the footpad of the right hind limb, mice were treated with IL-12 and/or GM-CSF administered intratumorally for 7 consecutive days. IL-12 used both at a high (1 µg) and at a low (0.01 µg) dose per day produced retardation of tumor growth, although neither treatment resulted in any significant prolongation of the survival of tumor-bearing mice. GM-CSF did not by itself exert antitumor activity in this model; however, it potentiated antitumor effects of IL-12. In particular, survival of tumor-bearing mice treated with IL-12 (0.01 µg per day) and GM-CSF was significantly prolonged compared with that in mice treated with either IL-12 or GM-CSF alone.

Absolute counts of peripheral blood leukocyte subpopulations in intraabdominal sepsis and pneumonia-derived sepsis: a pilot study

The leading pathophysiological changes during sepsis include systemic abnormalities in the immune response. Due to the general character of these disturbances, sepsis is usually studied as a homogenous clinical condition. We aimed to compare the immune response in intraabdominal sepsis (IAS) and pneumonia-derived sepsis (PDS). The following cell populations were examined: white blood cell count (WBC), monocytes, lymphocytes: CD3+, CD4+ and CD8+ T cells, B cells, and NK cells. In both studied groups (i.e. IAS and PDS), the WBC was elevated. However, it was significantly higher in the IAS group than in the PDS group. The difference was due to a lower granulocyte count, as well as a lower monocyte count in PDS. We found no significant correlation between the total lymphocyte number and CD3+CD8+ T cells in either form of sepsis. Similarly, we observed no correlation between the total lymphocyte number and the NK cells subset in IAS. However, the numbers of CD3+CD8+ and NK cells correlated similarly in both types of sepsis. Both studied types of sepsis induced profound lymphocytopenia, with marked loss of CD8+ T cells and the NK cells. However, the similar relation between them, which was independent of the infection type, suggests that the NK and CD3+CD8+ cells have shared mechanisms of regulation. The primary site of infection has an impact on the global immune reaction. These alternations include especially myeloid cells: granulocytes and monocytes which disappear from peripheral blood during PDS, but increase in IAS.

Myogenic Potential of Mesenchymal Stem Cells - the Case of Adhesive Fraction of Human Umbilical Cord Blood Cells

Different sources of stem cells are considered as a potential source of precursor cells that could improve skeletal muscle regeneration. Under physiological conditions muscle regeneration is based on the satellite cells, i.e. adult muscle precursor cells that are localized between muscle fiber and surrounding basal lamina. These cells remain quiescent but after skeletal muscle injury activate, proliferate, differentiate, and fuse either to form new muscle fibers or reconstruct the damaged ones. As it was shown in many studies few populations of stem cells other than satellite cells are able to support skeletal muscle regeneration. Among them are mesenchymal stem cells (MSCs) that are present in many niches within adult organism and also in fetal tissues, such as human umbilical cord blood (HUCB) or umbilical cord connective tissue, i.e. Whartons jelly. Thus, MSCs are intensively tested to prove that they are able to differentiate into various cell types, including skeletal myoblasts, and therefore could be useful in regenerative medicine. In our previous study we showed that MSCs isolated from Whartons jelly expressed pluripotency as well as myogenic markers and were able to undergo myogenic differentiation both in vitro and in vivo. We also analyzed the potential of HUCB cells population which contains not only MSCs but also hematopoietic precursors. Our analyses of whole population of HUCB cells showed that these cells express myogenic regulatory factors, i.e. MyoD, and are able to contribute to skeletal muscle regeneration. In the present study we document that adherent fraction of HUCB cells, i.e. the cells that constitute the subpopulation enriched in MSCs, expresses pluripotency and myogenic markers, and have a positive impact at the regeneration of injured mouse skeletal muscle.

Survival analysis of Escherichia coli encapsulated in a hollow fiber membrane in vitro and in vivo: preliminary report.

The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HFs space only.

The Cytotoxic Effect of Polyelectrolyte Shells Coated Bacterial Cells on Human Leukemia Cells

Encapsulation of cells in polymeric shells allowing for separation of biological material from produced factors may find application in the systems for biological processes regulation. Inadequate efficiency of existing therapeutic anticancer regiments and the rise of multi-drug resistant cancer cells have required investigations into novel anticancer strategies. Enhancement of apoptosis in tumors has been suggested as a new anticancer strategy. Pathogenic microorganisms may have the role as the source of agents for apoptotic therapy. Modified cells of Bacillus subtilis were encapsulated using layer-by-layer technique within polymeric shells for application in local anti-tumor therapy. The applied shells were modified with incorporated fullerene derivate to ensure the layers stability and integrity.The impact of modified nano-thin shells coated bacterial cells on human leukemia cells was evaluated in vitro. It was observed that coating with applied polyelectrolyte layers with incorporated fullerenol allowed for bacterial cells functioning during the culture period and the lethal impact on eukaryotic cells was observed. Applied membrane conformation allowing for functioning of encapsulated microorganisms may be recommended or coating shells for local anti-tumor treatment purposes.