Mesenchymal stem cells and their use in therapy: what has been achieved?

The considerable therapeutic potential of human multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) has generated increasing interest in a wide variety of biomedical disciplines. Nevertheless, researchers report studies on MSCs using different methods of isolation and expansion, as well as different approaches to characterize them; therefore, it is increasingly difficult to compare and contrast study outcomes. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed minimal criteria to define human MSCs. First, MSCs must be plastic-adherent when maintained in standard culture conditions (α minimal essential medium plus 20% fetal bovine serum). Second, MSCs must express CD105, CD73 and CD90, and MSCs must lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules. Third, MSCs must differentiate into osteoblasts, adipocytes and chondroblasts in vitro. MSCs are isolated from many adult tissues, in particular from bone marrow and adipose tissue. Along with their capacity to differentiate and transdifferentiate into cells of different lineages, these cells have also generated great interest for their ability to display immunomodulatory capacities. Indeed, a major breakthrough was the finding that MSCs are able to induce peripheral tolerance, suggesting that they may be used as therapeutic tools in immune-mediated disorders. Although no significant adverse events have been reported in clinical trials to date, all interventional therapies have some inherent risks. Potential risks for undesirable events, such as tumor development, that might occur while using these stem cells for therapy must be taken into account and contrasted against the potential benefits to patients.

Oligodeoxynucleotide IMT504: lack of effect on immune parameters during islet regeneration in single dose streptozotocin-induced diabetes.

BACKGROUND: We have shown that oligodeoxynucleotide IMT504 improved blood glucose and islet beta-cell content in streptozotocin (STZ)-induced diabetic rats, inducing early expression of progenitor markers. Here we determined the effect of IMT504 on islet infiltration and on immunomodulatory proteins indoleamine 2,3-dioxygenase (IDO) and TNF-α-stimulated gene/protein 6 (TSG-6) in islets of STZ-diabetic rats, at the time of progenitor markers expression. METHODS: Male rats were i.p. injected with STZ [60 mg/kg body weight (BW)] or citrate buffer (control) (day 1). Starting on day 4, STZ animals were daily treated with saline (STZ-saline) or IMT504 (20 mg/kg BW/day s.c., STZ-IMT504) and killed after two consecutive decreases in blood glucose. Islet area and insulin expression, CD3 (T lymphocytes), CD68 (macrophages), IDO and TSG-6 immunostainings were determined. Islet infiltration was also evaluated by haematoxylin staining. RESULTS: STZ-induced diabetes in rats, with an important decrease in islet area was reversed by IMT504. Diabetes development did not involve islet infiltration, determined by haematoxylin and by the absence of significant T lymphocyte and macrophage presence. IMT504 did not induce changes in these parameters. IDO was not expressed in controls; the percentages of IDO-positive islets were very low and similar in STZ-saline and STZ-IMT504. Scarce TSG-6 was expressed in all groups, without significant differences. CONCLUSIONS: IMT504 improved insulin content but did not alter IDO or TSG-6 staining in islets of STZ-diabetic rats, suggesting that they do not participate in the IMT504-induced repair process. IMT504 did not per se modify leukocyte presence in islets of diabetic rats.

Oligodeoxynucleotide IMT504 induces a marked recovery in a streptozotocin-induced model of diabetes in rats: correlation with an early increase in the expression of nestin and neurogenin 3 progenitor cell markers.

AIMS/HYPOTHESIS: IMT504 is an oligonucleotide that promotes tissue repair in bone injury and neuropathic pain models by stimulating progenitor cells. Here we evaluated the effect of IMT504 on the recovery of islet function in a streptozotocin (STZ)-induced model of diabetes in the rat. METHODS: Male Sprague-Dawley rats were injected with STZ (60 mg/kg, i.p., day 1) or citrate buffer (Control). Animals with glycaemia between 11 and 20 mmol/l on day 4 were injected with IMT504 (4 mg/animal in saline, s.c., STZ-IMT504) or with saline (STZ-Saline) for 10 days. Glycaemia and water and food intake were recorded for 33 days. Intraperitoneal glucose tolerance tests (IPGTTs) were performed on day 30. On day 35, overnight-fasted animals were killed and blood samples and pancreases collected for hormonal and histological studies. A second group of STZ-IMT504 rats was killed, together with Control and STZ-Saline rats, after two consecutive days of blood glucose decreases after the beginning of IMT504 treatment. Pancreases were collected and proliferating cell nuclear antigen (PCNA), nestin and neurogenin 3 (NGN3) detected by immunohistochemistry. RESULTS: IMT504 greatly improved blood glucose and food and water intakes in STZ-IMT504 rats by day 8, as well as IPGTTs on day 30. Significant increases in islet number and beta cell content were observed in STZ-IMT504 rats (day 33). Furthermore, after two to five IMT504 injections, blood glucose decreased, and an increase in pancreatic nestin (mainly in endothelial cells), PCNA and NGN3 production (in islets) was observed in STZ-IMT504 rats. CONCLUSIONS/INTERPRETATION: IMT504 induced a marked recovery of STZ-induced diabetes that correlated with early production of progenitor cell markers, such as nestin and NGN3.

Cardiomyogenic differentiation of human bone marrow mesenchymal cells: Role of cardiac extract from neonatal rat cardiomyocytes.

Bone marrow mesenchymal stromal cells (BM-MSCs) with regenerative potential have been identified in heart. Whether these cells become new cardiac lineage cells by phenomena of transdifferentiation or fusion is also being investigated. Although, these mechanisms give cardiomyocytes, it has to be considered that MSCs transplantation could carry out ossification and calcification processes. An alternative might be the use of myocytes; however, the problem is the arrythmia. For those reasons, is that we investigated how to obtain cardiomyocyte-like cells from human MSCs (hMSCs). The aim of the present work was to evaluate a nuclear reprogramming of the hMSCs by a neonatal rat cardiomyocytes extract (EX) using Streptolysin O (SLO) treatment. hMSCs treated with 57.5ng/ml SLO presented ball-like, stick-like and myotube-like morphology. In the absence of cardiomyogenic stimuli, hMSCs expressed markers of cardiac phenotype-like sarcomeric alpha-actinin, connexin-43 and GATA-4. However, when hMSCs were treated with SLO+EX or 10 microM of 5-azacytidine (5-AZA), the expression of these markers were significantly increased and furthermore, expressed SERCA-2, cardiac Troponin I, beta-MyHC, desmin, MLC-2a and MLC-2v thus showing the phenotype of mature cardiomyocytes. PCR analysis showed that cardiomyocyte-related genes, such as beta1-adrenergic receptor (beta1-AR), MLC-2a and cardiac Troponin T, were expressed after SLO+EX treatment like with 5-AZA. We concluded that the extract of neonatal rat cardiomyocytes could promote a nuclear modification of hMSCs to cardiomyogenic-like cells differentiation. Since the 5-AZA treatment appears to be genotoxic and taking into account the obtained results, the nuclear reprogramming by cell extract may be an approach leading to the identification of soluble factors that drives the reprogramming.

Interleukin-1 beta, transforming growth factor beta 1, prostaglandin E2, and fibronectin levels in the conditioned mediums of bone marrow fibroblast cultures from lung and breast cancer patients.

We analyzed the ability of the bone marrow (BM) stromal cells to achieve confluence and their proliferative capacity in BM primary cultures from 30 untreated lung cancer patients (LCP), 27 breast cancer patients (BCP), and 30 normal controls (NC) when these confluent cells were induced to proliferate following four continuous subcultures. Moreover, we evaluated the production of interleukin-1 beta (IL-1beta), transforming growth factor beta 1 (TGF-beta1), fibronectin, and prostaglandin E2 (PGE2) by pure fibroblasts (fourth passage). A fibroblast colony-forming units (CFU-F) assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta, TGF-beta1, and fibronectin in conditioned mediums (CM) of fibroblast cultures were measured by enzyme-linked immunosorbent assay (ELISA) kit and PGE(2) by radioimmunoassay (RIA) kit. Confluence was achieved in the 60% of LCP and 78% of BCP primary cultures compared with 100% of NC, and only fibroblasts from seven LCP and six BCP cultures had the capacity to proliferate following four subcultures. Levels of IL-1beta were below 10 pg/ml in both patient groups, while NC had a mean value of 5882.57+/-221.61 pg/ml. Levels of TGF-beta1 in BCP were lower than NC values ( P<0.05). LCP and BCP had significantly decreased levels of fibronectin when compared to NC values ( P<0.05 and P<0.01, respectively). Levels of PGE2 in LCP were higher compared to NC ( P<0.01). In conclusion, BM fibroblasts from LCP and BCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta, TGF-beta1, fibronectin, and PGE2 production.

Bone marrow fibroblasts in patients with advanced lung cancer

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1ß and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46 percent) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100 percent). Levels of spontaneously released IL-1ß were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 + or - 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 + or - 23.6 pg/ml) compared to NC (18.5 + or - 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1ß and PGE2 production

Bone marrow fibroblasts in patients with advanced lung cancer.

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1beta (IL-1beta) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46%) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100%). Levels of spontaneously released IL-1beta were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 +/- 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 +/- 23.6 pg/ml) compared to NC (18.5 +/- 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta and PGE2 production.

Bone marrow fibroblastic progenitors in patients with advanced breast cancer.

Bone marrow fibroblast colony-forming cells (CFU-F) were studied in fifteen consecutive untreated breast cancer patients (BCP) with clinical stages III and IV, and in sixteen normal controls (NC). A decreased number of CFU-F was observed in BCP compared to NC (p < 0.004). Confluence of the adherent cell layer was observed in all normal bone marrow mononuclear cells (MC) cultures, while a lower proportion of cultures from BCP (11/15) showed confluent adherent cell layers. When MC cultures of BCP were treated with indomethacin (Indo, 10(-6)M) 50% of them increased the number of CFU-F compared to the value obtained without treatment. In addition, a significant increase in the release of PGE2 in BCP cultures was observed before Indo treatment. Moreover, after MC were fractionated into adherent and non-adherent progenitors, the CFU-F decreased in both types of fractions of BCP compared to NC value (p < 0.02 and < 0.05, respectively). The number of light density MC per 10 ml of bone marrow aspirate and the number of trypsin-sensitive adherent progenitors were lower than NC in BCP (p < 0.02 and 0.013, respectively). Total MC and fibroblasts (fourth passage) were cultivated to evaluate the production of interleukin-1 beta (IL-1 beta) by ELISA methodology. Results indicated no difference of IL 1 beta spontaneous release when total MC cultures of both groups were compared. However, the levels of this cytokine were lower (< 10 pg/ml) in fibroblast culture supernatants of BCP compared to NC (1,217 +/- 74 pg/ml). Fibroblast cultures from BCP showed low or no release of IL-1 beta after muramyl-dipeptide (MDP. 1 microgram/ml) stimulation. In conclusion, the defective proliferative and confluence capacity of BCP fibroblastic progenitors may be related to the decrease in the production of IL-1 beta by these precursors.

Intracytoplasmatic and extracellular interleukin-1 production by monocytes of lung and colorectal cancer patients.

We studied the production of interleukin-1 (IL-1) by peripheral blood monocytes (Mo) from twelve normal subjects (NS) and eight and nine untreated lung and colorectal cancer patients (CP), respectively. No significant changes of extracellular IL-1 biological activity was observed between CP and NS by thymocyte proliferation assay. This result was independent that the cells were treated or not with lipopolisaccharide from E. coli (LPS, 10 micrograms/ml). Moreover, CP present normal amount of antigenic IL-1 beta in LPS treated Mo culture supernatants by enzyme-linked immunosorbent assay (ELISA). The biological activity of IL-1 released was not significant modified after indomethacin (Indo, 10(-6)M) and LPS + Indo treatments. Furthermore, patients showed a low percentage of LPS activated Mo with intracytoplasmatic IL-1 (alpha + beta) compared to normal values. These results were obtained by immuno-alkaline phosphatase staining using monoclonal antibody anti IL-1 (alpha + beta). In conclusion, CP had a reduced number of Mo with intracytoplasmatic IL-1 (alpha + beta) and the difference observed may depend on degradation or in the rate of synthesis of this cytokine.

Effects of sarcoma 180 growth on interleukin-1 and circulating immune complexes.

Cultured splenic mononuclear adherent cells (SMAc) from normal BALB/c mice as well as those from mice bearing 10-day sarcoma 180 (S180), exhibited a marked increase in Escherichia coli lipopolysaccharide-stimulated interleukin-1 (IL-1) production, when compared to spontaneous values. On days 20 and 30 following S180 challenge, a decrease in this effect on IL-1 production in treated and untreated SMAc was observed. Concomitantly with the alterations in the regulation of IL-1 production during tumor growth, an increase in the levels of prostaglandin E2 and serum immune complexes could be detected. These data suggest that the immunosuppression associated with later stages of tumor development may be due to direct effects on monocytes, by means of a down-regulation of IL-1 production.

[Production of interleukin 1 during tumor growth]. / Producción de interleuquina 1 durante el crecimiento tumoral.

The production of IL-1 by splenic mononuclear adherent cells (MAC) from BALB/c mice inoculated with Sarcoma 180 (S180) was examined as a possible mechanism underlying the immunosuppression observed in tumor bearing mice. Two different inducers of IL-1 were used to stimulate MAC. A natural polysaccharide PCj3 (1, 5, 10 micrograms/ml) and a lipopolysaccharide (LPS) of E. coli (1, 5, 10, 20 micrograms/ml). The IL-1 activity was assayed by the capacity of the supernatants of MAC cultures in different dilutions (1/5, 1/10, 1/20) to enhance the mitogenic response of murine thymocytes to phytohemagglutinin (PHA). Both stimulants induced comparable levels of IL-1 in normal and 10 day tumor bearing mice. Normal MAC were able to elaborate IL-1 spontaneously in the presence of 1% FCS: the optimum LPS concentration was 20 micrograms/ml in the 1/5 dilution. With regard to PCj3, the optimum concentration was 5 and 10 micrograms/ml in the 1/5 dilution (p less than 0.001 vs control). The maximum activity of IL-1 for MAC of 10 day tumor bearing mice was given by the concentration of 5 micrograms/ml for both stimulants. When MAC were incubated with LPS, the IL-1 production was dose dependent while PCj3 seemed to have reached a saturation level between 5 and 10 micrograms/ml. The increase in tumor size (day 20-30) was associated with a significant decrease in IL-1 production by MAC in response to both stimulants. Therefore, the immunosuppression associated with the late stages of tumor growth may be due to inhibition of IL-1 production.

Producción de interleuquina 1 durante el crecimiento tumoral. / [Production of interleukin 1 during tumor growth]

The production of IL-1 by splenic mononuclear adherent cells (MAC) from BALB/c mice inoculated with Sarcoma 180 (S180) was examined as a possible mechanism underlying the immunosuppression observed in tumor bearing mice. Two different inducers of IL-1 were used to stimulate MAC. A natural polysaccharide PCj3 (1, 5, 10 micrograms/ml) and a lipopolysaccharide (LPS) of E. coli (1, 5, 10, 20 micrograms/ml). The IL-1 activity was assayed by the capacity of the supernatants of MAC cultures in different dilutions (1/5, 1/10, 1/20) to enhance the mitogenic response of murine thymocytes to phytohemagglutinin (PHA). Both stimulants induced comparable levels of IL-1 in normal and 10 day tumor bearing mice. Normal MAC were able to elaborate IL-1 spontaneously in the presence of 1

FCS: the optimum LPS concentration was 20 micrograms/ml in the 1/5 dilution. With regard to PCj3, the optimum concentration was 5 and 10 micrograms/ml in the 1/5 dilution (p less than 0.001 vs control). The maximum activity of IL-1 for MAC of 10 day tumor bearing mice was given by the concentration of 5 micrograms/ml for both stimulants. When MAC were incubated with LPS, the IL-1 production was dose dependent while PCj3 seemed to have reached a saturation level between 5 and 10 micrograms/ml. The increase in tumor size (day 20-30) was associated with a significant decrease in IL-1 production by MAC in response to both stimulants. Therefore, the immunosuppression associated with the late stages of tumor growth may be due to inhibition of IL-1 production.

Antitumoral activity of polysaccharide isolated from Cyttaria johowii on the growth of sarcoma 180 in normal and splenectomized mice.

The antitumoral activity of the polysaccharide (PCj3) isolated from the Cyttaria johowii fungus on the growth of solid Sarcoma 180 (S180) in normal and splenectomized BALB/c mice was studied, observing that this treatment inhibited tumor growth in normal and splenectomized mice. At the same time, the effect of PCj3 on peripheral blood leukocyte populations on the 8th, 15th and 25th day of the experiment was evaluated. Results obtained on day 8 showed that all groups inoculated with S180 suffered an increase in the number of lymphocytes and neutrophils, corresponding the greatest values to splenectomized tumor bearing mice treated with PCj3. Neutrophils increase continued in all animals even without PCj3 treatment until the end of the experiment, while lymphocytosis was only maintained in the splenectomized groups. There was an increase in the number of monocytes on day 8 caused by PCj3 treatment with respect to normal values. It was concluded that PCj3 treatment more effectively delayed S180 growth in splenectomized mice, increasing the survival days as well as the lymphocytes, neutrophils and monocytes values on the 8th day of tumor growth with respect to the other groups.

Antitumoral activity of polysaccharide isolated from Cyttaria johowii on the growth of sarcoma 180 in normal and splenectomized mice.

The antitumoral activity of the polysaccharide (PCj3) isolated from the Cyttaria johowii fungus on the growth of solid Sarcoma 180 (S180) in normal and splenectomized BALB/c mice was studied, observing that this treatment inhibited tumor growth in normal and splenectomized mice. At the same time, the effect of PCj3 on peripheral blood leukocyte populations on the 8th, 15th and 25th day of the experiment was evaluated. Results obtained on day 8 showed that all groups inoculated with S180 suffered an increase in the number of lymphocytes and neutrophils, corresponding the greatest values to splenectomized tumor bearing mice treated with PCj3. Neutrophils increase continued in all animals even without PCj3 treatment until the end of the experiment, while lymphocytosis was only maintained in the splenectomized groups. There was an increase in the number of monocytes on day 8 caused by PCj3 treatment with respect to normal values. It was concluded that PCj3 treatment more effectively delayed S180 growth in splenectomized mice, increasing the survival days as well as the lymphocytes, neutrophils and monocytes values on the 8th day of tumor growth with respect to the other groups.

Circulating immune complexes and peripheral B lymphocytes in non insulin dependent diabetic patients.

Serum circulating immune complexes (CIC) were measured in 27 patients with non insulin dependent diabetes (NIDD). This was done by measuring the degree of binding to human red blood cells by the C3b complement fraction. At the same time, the percentage of B lymphocytes in peripheral blood was evaluated by means of the direct immunofluorescence technique for surface IgG and EAC rosettes for cells with receptors to C3b complement fraction. Twenty normal control subjects were simultaneously studied by the same methodology. An increase in serum CIC was observed in NIDD patients, as compared to healthy subjects. Values were 35.8 +/- 3.2 and 25.6 +/- 2.1 micrograms/ml, respectively. The percentage of cells with surface IgG was 10.2 +/- 0.8 in diabetic patients; this value was significantly higher than that found in the control group (6.0 +/- 0.8). No significant quantitative difference in the percentage of EAC binding cells was found between NIDD patients and the control group. When NIDD patients were divided into two groups, those with and those without microvascular complications, neither differences in CIC levels nor in the percentage of B lymphocytes were found. Nor any correlation could be found between the highest individual CIC levels and the highest percentage of lymphocytes with surface IgG. These data show an increase of CIC levels and of cells with surface IgG in NIDD patients who had not received insulin at least not in a constant or prolonged therapy. This could allow us to suspect the existence of antigen-antibody complexes different to insulin-antiinsulin CIC found in insulin dependent diabetes.

Circulating immune complexes and peripheral B lymphocytes in non insulin dependent diabetic patients.

Serum circulating immune complexes (CIC) were measured in 27 patients with non insulin dependent diabetes (NIDD). This was done by measuring the degree of binding to human red blood cells by the C3b complement fraction. At the same time, the percentage of B lymphocytes in peripheral blood was evaluated by means of the direct immunofluorescence technique for surface IgG and EAC rosettes for cells with receptors to C3b complement fraction. Twenty normal control subjects were simultaneously studied by the same methodology. An increase in serum CIC was observed in NIDD patients, as compared to healthy subjects. Values were 35.8 +/- 3.2 and 25.6 +/- 2.1 micrograms/ml, respectively. The percentage of cells with surface IgG was 10.2 +/- 0.8 in diabetic patients; this value was significantly higher than that found in the control group (6.0 +/- 0.8). No significant quantitative difference in the percentage of EAC binding cells was found between NIDD patients and the control group. When NIDD patients were divided into two groups, those with and those without microvascular complications, neither differences in CIC levels nor in the percentage of B lymphocytes were found. Nor any correlation could be found between the highest individual CIC levels and the highest percentage of lymphocytes with surface IgG. These data show an increase of CIC levels and of cells with surface IgG in NIDD patients who had not received insulin at least not in a constant or prolonged therapy. This could allow us to suspect the existence of antigen-antibody complexes different to insulin-antiinsulin CIC found in insulin dependent diabetes.