Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium.

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable "off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe, animal product-free medium containing 2%, 5%, and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery, viability, apoptosis, proliferation rate, expression of a broad panel of MSC markers, and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO, respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO, respectively, were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity, ALP surface expression and Ca⁺⁺ deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution, also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery, respectively, less than 10% of apoptotic cells, and normal proliferation, marker expression, and osteogenic potential. Overall, our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents.

Markers distinguishing mesenchymal stem cells from fibroblasts are downregulated with passaging.

Expansion of plastic-adherent bone marrow-derived mesenchymal stem cells (MSCs) results in gradual loss of osteogenic potential after passage 5-6. One explanation is contamination of MSC cultures with mature cells including fibroblasts. Identification and elimination of fibroblasts from MSC cultures could improve MSC yield and differentiation potential and also prevent tumor formation after MSC transplantation. However, no specific markers currently exist that can reliably discriminate between MSCs and fibroblasts. Flow cytometry analysis demonstrated that markers currently used to define MSCs, such as CD105, CD166, CD90, CD44, CD29, CD73, and CD9, are also expressed on human skin or lung fibroblasts. However, the level of expression of CD166 was significantly higher and that of CD9 was significantly lower in MSCs than in fibroblasts. CD146 was expressed only in MSCs. Using small focused microarrays, new markers differentially expressed in MSCs and fibroblasts were identified. Real-time polymerase chain reaction confirmed that expression of CD106, integrin alpha 11, and insulin-like growth factor-2 in MSCs was at least 10-fold higher than in fibroblasts; whereas expression of matrix metalloproteinase 1 and matrix metalloproteinase 3 was almost 100-fold lower. Flow cytometry and immunostaining demonstrated that CD106 protein expression on cell surface could be upregulated in MSCs but not in fibroblasts by the treatment with tumor necrosis factor-alpha. Comparison of surface expression of commonly used and newly identified MSC markers in MSCs cultures of passage 2 and passage 6 demonstrated that CD106 (with and without tumor necrosis factor-alpha treatment), integrin alpha 11, and CD146 were downregulated in MSCs of passage 6, and CD9 was upregulated; whereas all other markers did not change. Newly identified markers that have robust differences of expression in MSCs and fibroblasts on gene and protein level could be used for quality control of MSC cultures after expansion, cryopreservation, gene transfection, and other manipulations.

Proteasomal degradation regulates expression of porphobilinogen deaminase (PBGD) mutants of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a neuropathic disease caused by a dominant inherited deficiency in porphobilinogen deaminase (PBGD). We investigated the expression and the degradation of the human PBGD-mutations G748A, G748C and 887insA following transfection into human SH-SY5Y neuroblastoma cells. Mutant proteins exhibited reduced protein expression compared to transfected wild-type (wt) PBGD as revealed by Western blotting. The transcription levels assessed by real-time PCR of these mutant species were identical to those of the wild type. Immuno-fluorescence microscopy revealed reduced cellular distribution of the mutated PBGDs in the cytosol and the nucleus in comparison to the wild-type PBGD. Enhanced cellular accumulation of the mutated and wild-type PBGDs was detected following inhibition of the proteasome by the inhibitors CLBL and hemin. Elevated expression of wt and mutated PBGD protein levels was either achieved by hemin or heme-arginate treatment. On the other hand, enhanced PBGD degradation was achieved by lead poisoning of ALAD in the SH-SY5Y cells concomitant with acceleration of proteasomal activity, most probably by ALAD participation in proteasomal regulation [G.G. Guo, M. Gu, J.D. Etlinger, 240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. J Biol Chem 1994; 269:12399-402.] Our results suggest that the difference in expression between the wild-type and mutant proteins appears to be regulated on the level of protein degradation. In conclusion, we demonstrate that the PBGD cellular pool is controlled by the proteasome activity, which in turn is down regulated by hemin or up-regulated by Pb-ALAD.

Regulation of porphyrin synthesis and photodynamic therapy in heavy metal intoxication.

Protoporphyrin IX (PpIX) synthesis by malignant cells is successfully exploited for photodynamic therapy (PDT) following administration of 5-aminolevulinic acid (ALA) and light irradiation. The influence of two environmental heavy metal poisons, lead and gallium, on PpIX-synthesis and ALA-PDT was studied in two neu-ronal cell lines, SH-SY5Y neuroblastoma and PC12 pheochromocytoma. The heavy metal intoxication affected two of the heme-synthesis enzymes, ALA-dehydratase (ALAD) and porphobilinogen deaminase (PBGD). The present results show that lead poisoning significantly decreased the PBGD cellular level and inhibited its enzymatic activity, whereas the effects of gallium were less prominent. Although, the protein levels were reduced, the mRNA levels of PBGD remained unchanged during metal intoxication. These findings show additional inhibitory activity of lead on top of its classical effect on ALAD. Proteasome activity was enhanced during lead treatment, as measured by the AMC fluorigenic proteasome assay. The reduction in PBGD levels was not a consequence of PBGD mRNA reduced synthesis, which remained unchanged as shown by RT-PCR analysis. As a result of the lead poisoning, marked alterations in the cell cycle were observed, including a decreased G1 phase and an increased number of S phase cells. The efficacy of ALA-PDT was reduced in correlation with decreased activities of the enzymes during lead intoxication. We may conclude that lead poisoning adversely affects the outcome of ALA photodynamic therapy of cancer.

Increased expression of the hPim-2 gene in human chronic lymphocytic leukemia and non-Hodgkin lymphoma.

Pim-1 and Pim-2 are murine proto-oncogenes implicated in lymphomagenesis. The aim of this study was to investigate whether the human Pim-2 (hPim-2) expression is altered in chronic lymphocytic leukemia (B-CLL) and non-Hodgkin's lymphomas (NHL). We analyzed hPim-2 expression in 48 patients with NHL and CLL by quantitative in-situ hybridization, quantitative RT-PCR and FACS analysis. In-situ hybridization revealed a 5.5 +/- 2.2 times higher expression of hPim-2 in NHL over normal lymphocytes (P < 0.001). Similarly, with quantitative RT-PCR, expression in NHL was 1.5 to 2.6 times higher in involved splenic foci compared to nearby uninvolved regions (n = 3). hPim-2 mRNA was increased 3-folds in B-CLL over normal B-cells (P < 0.006). The increased hPim-2 levels correlated with lymphocyte doubling time (DT), in that mRNA levels were two times greater in patients with rapid DT (P < 0.006). Moreover, a significant correlation was found between hPim-2 expression and the Binet staging system of CLL (P < 0.022). The hPIM-2-protein expression was also upregulated in CLL, as assessed by FACS analysis. Therefore, this report provides direct evidence for a linkage of hPim-2 upregulation to NHL and CLL in man. This relationship between hPim-2 and NHL and CLL raises a number of novel mechanistic options for the genesis and/or progression of some types of human lymphomas.