Xenogeneic protein-free cultivation of mesenchymal stromal cells - towards clinical applications.

We have studied a rapid cultivation method for human mesenchymal stromal cells based on CellGroTM medium and human serum, supplemented with insulin, ascorbic acid, dexamethasone, epidermal growth factor, platelet-derived growth factor BB, macrophage colony-stimulating factor and fibroblast growth factor 2. This study has shown that rapid expansion of human multipotent mesenchymal stromal cells using human serum could not be achieved without addition of growth factors. Furthermore, we have found that insulin and, quite probably, epidermal growth factor may be omitted from our formula without loss of colony-forming capacity or total cell yield. On the other hand, dexamethasone, ascorbic acid and fibroblast growth factor 2 were necessary for the growth and colony-forming capacity of multipotent mesenchymal stromal cells, while platelet-derived growth factor BB prevented their differentiation into adipogenic lineage. Moreover, multipotent mesenchymal stromal cells cultivated in our system expressed higher levels of bone morphogenetic protein 2, but not bone morphogenetic protein 7, than cells cultivated in α-MEM with foetal bovine serum. This shows that our system promotes differentiation of mesenchymal cells towards osteogenic and chondrogenic lineages, making them more suitable for bone and cartilage engineering than cells grown in conventional media. Furthermore, we have proved that these cells may be conveniently cultivated in a closed system, in vessels certified for clinical use (RoboFlaskTM), making the transfer of our cultivation technology to good clinical practice easier and more convenient.

Circulating endothelial precursor cells (EPC) in patients undergoing allogeneic haematopoietic progenitor cell transplantation.

We have studied the number of endothelial precursor cells in eighteen patients undergoing allogeneic haematopoietic stem cell transplantation. Endothelial precursor cells were evaluated by colony-forming assay and compared to healthy controls. Patients undergoing allogeneic haematopoietic stem cell transplantation had significantly lower numbers of endothelial precursor cells before the procedure than healthy controls. The numbers of endothelial precursor cells were even lower in the first year after the treatment and seemed to recover partially after twelve months, but even then, they were lower than in healthy volunteers. On the other hand, the number of circulating CD146+CD31+ mature endothelial cells were higher than in healthy controls after more than a one-year follow-up. We hypothesize that lower numbers of endothelial precursor cells and higher numbers of endothelial cells in patients undergoing allogeneic haematopoietic stem cell transplantation reflect ongoing endothelial damage, probably caused by immunological mechanisms, and that this longterm damage may explain the higher risk of cardiovascular events in allogeneic haematopoietic stem cell transplant survivors.

Phenotypic characterization of human keratinocytes in coculture reveals differential effects of fibroblasts from benign fibrous histiocytoma (dermatofibroma) as compared to cells from its malignant form and to normal fibroblasts.

BACKGROUND: Benign and malignant fibrous histiocytoma present with a considerable difference concerning cellular organization in their vicinity. OBJECTIVE: Normally appearing epithelium covers the malignant form in contrast to hyperplastic epidermis for benign tumors. It is an open question as to whether the tumor-associated fibroblasts are capable to affect phenotypic features of normal keratinocytes, prompting this comparative analysis. METHODS: Fibroblasts were isolated from benign and malignant fibrous histiocytomas, respectively, and also from normal dermis. The resulting cell populations were thoroughly characterized immunocytochemically using a large panel of antibodies. The three fibroblast preparations were cocultured with normal interfollicular keratinocytes. Their phenotype was characterized for distinct properties including differentiation and proliferation. RESULTS: Fibroblasts prepared from both tumor types were phenotypically practically identical with normal dermal fibroblasts. Their activities on keratinocytes were different. Cells prepared from benign fibrous histiocytoma were capable to effect strong expression of keratin 19 and production of a galectin-1-rich extracellular matrix. Fibroblasts isolated from malignant fibrous histiocytoma led to a phenotype very similar to that when keratinocytes were cocultured with normal dermal fibroblasts. CONCLUSION: Fibroblasts prepared from benign fibrous histiocytoma were biologically active on keratinocytes in a particular manner. Our results on fibroblast activity are suggested to be relevant for morphologic differences observed in vivo between normal epidermis and epidermis adjacent to the studied tumor types.

Autologous intra-arterial infusion of bone marrow mononuclear cells in patients with critical leg ischemia.

AIM: The injection of bone marrow mononuclear cells (BMMC) into the gastrocnemius muscle has given promising results in patients with critical limb ischemia (CLI). In this article, we have assessed whether a less invasive procedure, i.e. intravascular BMMC infusion, could be effective in this population of patients. METHODS: A total of 28 limbs in 24 patients with CLI were treated. An amount of 276-700 mL of marrow blood was harvested from posterior iliac crests and BMMC were obtained by standard procedure used for bone marrow transplantation. After performance of digital subtraction angiography, BMMC were injected laterally through a 4 Fr sheet. Primary outcome was efficacy of the procedure measured as healing of defects, frequency of high amputations and change of ischemia grade; among secondary outcomes were safety of the procedure, angiographic changes and changes in quality of life. RESULTS: One year after treatment, all patients were alive and only 2 patients have undergone high amputation. Eleven of 14 defects have healed (78%) and Fontaine grade of ischemia has changed from median grade 3.5 to median grade 2 (P<0.0001). Collateral vessel development has improved by mean 1.13 and 1.3 points on a four-point semiquantitative scale in calf and foot, respectively (P<0.0001). There were no grade III-IV adverse events. According to the SF-36 quality of life questionnaire, 1 year after the procedure patients have reported significant improvement in all measured items. CONCLUSION: Intra-arterial infusion of BMMC can lead to significant and long-lasting subjective and objective improvements in patients with CLI. The results merit validation by randomized controlled studies in patients with less critical limb ischemia.

Circulating endothelial cells and circulating endothelial progenitors in kidney disease--victims, witnesses, or accomplices?

Nephrologists deal with a host of pathologic conditions involving renal and systemic endothelium. Both in native and transplanted kidneys, often the insult to the renal endothelium initiates the pathogenic process ultimately leading to the loss of organ function. Also, systemic atherosclerosis is accelerated in patients with renal dysfunction. In this review we would like to cover the possible role of CECs and their counterparts--circulating EPCs in the pathogenesis of endothelial dysfunction associated with chronic renal failure, ANCA-associated vasculitis, and progression of chronic renal disease.

Dendritic cells and T lymphocyte interactions in patients with lymphoid malignancies.

Dendritic cell (DC) vaccination is an attractive approach to the treatment of patients with lymphoid tumors. To evaluate its feasibility, we have tested the functional properties of DC and T-lymphocytes in patients with treated and untreated chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). Healthy volunteers were used both as controls and as a source of cells for allogeneic mixed leukocyte reaction (MLR). In these reactions, dendritic cells from both untreated and treated patients were comparable to dendritic cells from healthy volunteers. In all the untreated patients studied, autologous dendritic cells promoted the survival and proliferation of both CD4 and CD8 lymphocytes (though the proliferation response was much better in the CD4 subset), whereas only 3 out of 5 treated patients were able to mount this response with CD4 lymphocytes and 4 out of 5 with CD8 lymphocytes. In 3 out of 5 untreated patients, pulsing of DCs with tetanus toxoid promoted a better CD4 response than was achieved with unpulsed DCs, while none of 5 treated patients had an additional response after pulsing with tetanus toxoid. None of patients studied, either treated or untreated, had a better CD8 response to pulsed DCs than to unpulsed ones. During CD4 lymphocyte proliferation, more CD4(+)CD25(hi) lymphocytes were generated in both treated and untreated patients than in healthy controls. Poor proliferation of cytotoxic cells and preferential proliferation of CD4(+)CD25(hi) T-regulatory cells in response to self and/or foreign antigens might be one of the mechanisms responsible for immunosuppression and impaired tumor surveillance in patients with lymphoid malignancies.

Stromal fibroblasts from basal cell carcinoma affect phenotype of normal keratinocytes.

BACKGROUND: Epithelial-mesenchymal interactions are important not only to direct the course of prenatal development of skin and its appendages but also to influence the behaviour of transformed epithelial cells. OBJECTIVES: Evaluation of the role of stromal fibroblasts on the phenotype of epithelial cells of basal cell carcinoma (BCC). METHODS: The phenotype of human BCC was compared with the in vitro model where the growth and phenotypic pattern of normal human keratinocytes were monitored in co-culture with fibroblasts prepared from stroma of BCC (BCCFs), with normal dermal fibroblasts or with two established fibroblast lines. We visualized the expression of a panel of keratins, three types of endogenous lectin [galectin (Gal)-1, Gal-3 and Gal-7], binding sites for Gal-1 and Gal-3, a proliferation marker Ki67, nucleolar protein nucleostemin (NuclS) and membrane protein Ber-EP4. A phenotype and karyotype of BCCFs were also monitored. BCCFs were also grafted to NOD/LtSz-Rag1(null) mice to evaluate their malignant potential. RESULTS: Prolonged cultivation of BCCFs has led to morphological changes, loss of contact inhibition, loss of fibroblast surface antigens and progressive aneuploidity. However, a fully malignant phenotype did not develop as these cells did not form tumours in immunodeficient mice. Co-culture of BCCFs with normal keratinocytes in vitro led to their phenotypical changes resembling those in BCC, namely, expression of keratin 19. These keratinocytes also strongly express nuclear binding sites for Gal-1 and NuclS. This phenotype was not observed when normal keratinocytes were cultured with nontumour-originated fibroblasts. CONCLUSIONS: These observations indicate that BCCFs may differ from normal fibroblasts and may play a regulatory role in BCC biology.