Aparecida Maria Fontes

Mechanisms involved in the therapeutic properties of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been described as being able to give rise to several quite different mesenchymal cell phenotypes. However, the ability to differentiate is not the only characteristic that makes these cells attractive for therapeutic purposes. The secretion of a broad range of bioactive molecules by MSCs, such as growth factors, cytokines and chemokines, constitutes their most biologically significant role under injury conditions. Understanding this intricate secretory activity as well as the properties of MSCs in vivo is central to harnessing their clinical potential. Herein, we identify some of the molecules involved in the paracrine effects of MSCs with a perspective that these cells intrinsically belong to a perivascular niche in vivo, and discuss how this knowledge could be advantageously used in clinical applications.

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts.

OBJECTIVE The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.

Human hepatic stellate cell line (LX-2) exhibits characteristics of bone marrow-derived mesenchymal stem cells

The LX-2 cell line has characteristics of hepatic stellate cells (HSCs), which are considered pericytes of the hepatic microcirculatory system. Recent studies have suggested that HSCs might have mesenchymal origin. We have performed an extensive characterization of the LX-2 cells and have compared their features with those of mesenchymal cells. Our data show that LX-2 cells have a phenotype resembling activated HSCs as well as bone marrow-derived mesenchymal stem cells (BM-MSCs). Our immunophenotypic analysis showed that LX-2 cells are positive for activated HSC markers (αSMA, GFAP, nestin and CD271) and classical mesenchymal makers (CD105, CD44, CD29, CD13, CD90, HLA class-I, CD73, CD49e, CD166 and CD146) but negative for the endothelial marker CD31 and endothelial progenitor cell marker CD133 as well as hematopoietic markers (CD45 and CD34). LX-2 cells also express the same transcripts found in immortalized and primary BM-MSCs (vimentin, annexin 5, collagen 1A, NG2 and CD140b), although at different levels. We show that LX-2 cells are capable to differentiate into multilineage mesenchymal cells in vitro and can stimulate new blood vessel formation in vivo. LX-2 cells appear not to possess tumorigenic potential. Thus, the LX-2 cell line behaves as a multipotent cell line with similarity to BM-MSCs. This line should be useful for further studies to elucidate liver regeneration mechanisms and be the foundation for development of hepatic cell-based therapies.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, Sciaridae)

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly α-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5′ end, and beyond the 3′ end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier-based system.

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ∼4.82 ± 1.18 × 105 cell mL−1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy.

Effects of high-dose chemotherapy on bone marrow multipotent mesenchymal stromal cells isolated from lymphoma patients

OBJECTIVE High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT. MATERIALS AND METHODS We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing alpha-minimum essential medium plus 15% fetal bovine serum. RESULTS The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkins disease and non-Hodgkins lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group. CONCLUSIONS Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.

Knops blood group haplotypes among distinct Brazilian populations

BACKGROUND: The Knops blood group system consists of antigens encoded by exon 29 of complement receptor 1 (CR1) gene. To better elucidate the complexity of Knops group system, the frequency of six single‐nucleotide polymorphisms (SNPs) in three Brazilian populations is determined.

Differential expression of AURKA and AURKB genes in bone marrow stromal mesenchymal cells of myelodysplastic syndrome: correlation with G-banding analysis and FISH.

It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells.

Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells

P140K-MGMT and G156A-MGMT genes encode two O6-benzylguanine–resistant O6-alkylguanine DNA alkyltransferase proteins that confer a high degree of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O6-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O6-benzylguanine–resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O6-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED50 for O6-benzylguanine, 60 μmol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED50 for benzylguanine, >1,000 μmol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O6-benzylguanine (800 μmol/L) and temozolomide (1,000 μmol/L) or BCNU (20 μmol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O6-benzylguanine–resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection. [Mol Cancer Ther 2006;5(1):121–8]

Stable and high‐level production of recombinant Factor IX in human hepatic cell line

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high‐cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed‐phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.