HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like...     

Will transplantation of peripheral blood stem haematopoietic cells definitively replace bone marrow transplantation?

Findings assembled for a long time, practically from the end of the Second World War along with modern technology of genetic engineering which brought mass production of growth factors led at the beginning of the nineties of the 20th century to a rapid rise of transplantations of peripheral stem cells. Without exaggeration it may be said that the last decade of the 20th century is the decade of transplantations of peripheral stem cells. Peripheral stem cells comprise a wide range of haematopoietic cells incl. stem cells which are after a stimulus (antitumour chemotherapy or growth factors) released from bone marrow in high concentrations into the peripheral blood stream from where they can be obtained very simply by modern blood cell separators. They are suitable for autologous as well as allogenic transplantations. In autologous applications they have almost replaced bone marrow. Restoration of haematopoiesis is after their use more rapid as the transplant is richer. As regards allogenic application caution is still apparent, although even their ratio is very significant. However a higher rate of reactions of the graft to the host is feared. It seems however that this fear is not justified and that the richer transplant of peripheral stem cells can ensure a higher anti-tumourous effect of the transplantation. Peripheral stem cells are also used successfully in transplantations after so-called non-myeloablative regimes. It is beyond doubt that the use of peripheral stem cells will increase further. We may expect application of other growth factors with a higher mobilizing capacity. Peripheral stem cells will be subjected to further modifications in vitro. It will be also possible to increase their amount. A rich transplant makes it also possible to use it in transplantations from haploidentic donors. No doubt stem cell suspensions will become the objective of gene therapy.     

Stem cells and cancer： Evidence for bone marrow stem cells in epithelial cancers

Cancer commonly arises at the sites of chronic inflam-mation and infection.Although this association has longbeen recognized,the reason has remained unclear.With-in the gastrointestinal tract,there are many examplesof inflammatory conditions associated with cancer,andthese include reflux disease and Barrett's adenocarcino-ma of the esophagus,Helicobacter infection and gastriccancer,inflammatory bowel disease and colorectal cancerand viral hepatitis leading to hepatocellular carcinoma.There are several mechanisms by which chronic inflam-mation has been postulated to lead to cancer whichincludes enhanced proliferation in an endless attempt toheal damage,the presence of a persistent inflammatoryenvironment creating a pro-carcinogenic environmentand more recently a role for engraftment of circulatingmarrow-derived stem cells which may contribute to thestromal components of the tumor as well as the tumormass itself.Here we review the recent advances in ourunderstanding of the contributions of circulating bonemarrow-derived stem cells to the formation of tumors inanimal models as well as in human beings.     

Interferon-γ mediates the immunosuppression of bone marrow mesenchymal stem cells on T-lymphocytes in vitro

Objectives: In the present study, we first confirmed the suppressive function of MSCs in allogeneic T cell proliferation and then examined the underlying mechanisms for MSCs’ immunomodulation and the role of the pro-in?ammatory cytokine interferon (IFN)-γ.

Methods: Human MSCs were cultured in the presence or absence of IFN-γ. The expression level of prostaglandin E₂ (PGE₂), hepatocyte growth factor (HGF), transforming growth factor (TGF)-β₁ and indoleamine 2,3-dioxygenase (IDO) by MSCs were measured. T lymphocytes were isolated from peripheral blood of healthy donors and then induced to proliferate under the stimulation of anti-human CD3 mAb and anti-human CD28 mAb. In the presence of MSCs, T cell proliferation was examined by BrdU incorporation. In addition, PGE₂, HGF, TGF-β₁, Kynurenine, recombinant human IFN-γ and anti-IFN-γ mAb were added and cell proliferation was examined.

Results: Compared to the controls (MSCs alone), MSCs cocultured with IFN-γ expressed significantly higher concentrations of PGE₂, HGF and TGF-β₁. The mRNA level of IDO was remarkably increased. Human bone marrow-derived MSCs alone notably suppressed T lymphocytes proliferation in vitro. Addition of exogenous IFN-γ did not ablate the immunosuppressive effects of MSCs. Addition of anti-IFN-γ mAb partially restored suppression of T cell proliferation by MSCs.

Conclusions: Human MSCs constitutively expressed immunosuppressive levels of PGE₂, HGF and TGF-β₁. The proinflammatory cytokine IFN-γ exhibited synergistic effects with MSCs on immunosuppression, possibly by up-regulating PGE₂, HGF and TGF-β₁ in MSCs and inducting MSCs expression of IDO, involved in tryptophan catabolism.     

Expression of interferon-γ and tumor necrosis factor-α in bone marrow T cells and their levels in bone marrow plasma in patients with aplastic anemia

Immune-mediated stem cell damage has been postulated to be responsible for disease initiation and progression in aplastic anemia (AA). It is hypothesized that T lymphocytes play a major role in destroying the bone marrow (BM) stem cells of AA patients by infiltrating the BM and secreting excessive levels of anti-hematopoietic cytokines, interferon-gamma (IFN-), and tumor necrosis factor-alpha (TNF-). We undertook this study to assess the pathogenic significance of anti-hematopoietic cytokines such as IFN- and TNF- in BM T cells and plasma of AA patients. Significantly elevated levels of IFN- and TNF- were found in the BM plasma of AA patients compared to controls (p=0.05 and 0.006, respectively). Intracellular IFN- and not TNF- in BM CD3⁺ T cells of AA patients was significantly higher compared to controls (p=0.04 and p=0.2, respectively). A follow-up analysis of expression of these cytokines in BM T cells and their levels in BM plasma in five AA patients before and 180 days (6 months) after antithymocyte globulin (ATG) and cyclosporin A (CsA) therapy showed a decline 180 days after therapy compared to pre-therapy. We thus conclude that increased production of both IFN- and TNF- in the BM may contribute to disease pathogenesis in AA and ATG therapy may induce hematological remission by suppressing the elevated levels of IFN- and TNF- in AA BM.     

Generation of functional hepatocyte-like cells from human bone marrow mesenchymal stem cells by overexpression of transcription factor HNF4α and FOXA2

Background: Our previous study showed that overexpression of hepatocyte nuclear factor 4α(HNF4α) could directly promote mesenchymal stem cells(MSCs) to differentiate into hepatocyte-like cells. However, the efficiency of hepatic differentiation remains low. The purpose of our study was to establish an MSC cell line that overexpressed HNF4α and FOXA2 genes to obtain an increased hepatic differentiation efficiency and hepatocyte-like cells with more mature hepatocyte functions. Methods: Successful establishment of high-level HNF4α and FOXA2 co-overexpression in human induced hepatocyte-like cells(hi Hep cells) was verified by flow cytometry, immunofluorescence and RT-PCR. Measurements of albumin(ALB), urea, glucose, indocyanine green(ICG) uptake and release, cytochrome P450(CYP) activity and gene expression were used to analyze mature hepatic functions of hi Hep cells. Results: hi Hep cells efficiently express HNF4α and FOXA2 genes and proteins, exhibit typical epithelial morphology and acquire mature hepatocyte-like cell functions, including ALB secretion, urea production, ICG uptake and release, and glycogen storage. hi Hep cells can be activated by CYP inducers. The percentage of both ALB and α-1-antitrypsin(AAT)-positive cells was approximately 72.6%. The expression levels of hepatocyte-specific genes( ALB, AAT, and CYP1A1) and liver drug transport-related genes( ABCB1, ABCG2, and SLC22A18) in hi Hep cells were significantly higher than those in MSCs-Vector cells. The hi Hep cells did not form tumors after subcutaneous xenograft in BALB/c nude mice after 2 months. Conclusion: This study provides an accessible, feasible and efficient strategy to generate hi Hep cells from MSCs.     

Effect of interleukin-3, stem cell factor and granulocyte–macrophage colony-stimulating factor on committed stem cells, long-term culture initiating cells and bone marrow stroma in a one-step long-term bone marrow culture

Received: May 26, 1999 / Accepted: October 8, 1999     

Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation?

OBJECTIVE: The potential use of bone marrow stromal cells (MSCs) as a cellular therapy for chronic cardiac diseases relies on the ability of the cell to replicate extensively in vitro and to give rise to myogenic cells that can replace the damaged cardiomyocytes. For this reason the present study investigated the replication lifespan and chemical-induced cardiomyogenic differentiation of rat MSCs in vitro. METHODS: The primary and the successively passaged Wistar rat MSCs were exposed to different concentrations (3, 5 and 10 microM) of 5-azacytidine using different methods (single- or repeat-treatment). The growth properties and the fate of the cells were compared to their untreated counterparts by cell counting, immunocytochemistry and Western analysis. RESULTS: When seeded at a density of 2845 cells/cm(2) and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. When treated with 5-azacytidine for 24 h at day 3 of primary culture and the first subculture, the growth properties of the MSCs were not obviously affected. Neither the spontaneously beating cells nor the formation of myotubes were found in the primary and first passaged MSCs after a single treatment with 5-azacytidine and in cultures which underwent repeated 5-azacytidine-treatments during continuous subculturing to passage 2. The expressions of cardiac troponin I, cardiac myosin heavy chain and connexin 43 by the 5-azacytidine-treated MSCs were also undetectable at both immunocytochemistry and Western blot levels. The specificity and reliability of the detection methods were technically confirmed with cultured rat cardiomyocytes. CONCLUSIONS: Rat MSCs cannot be extensively expanded in vitro or be induced to differentiate in an expected cardiomyogenic way by 5-azacytidine-treatment, if the cells are not immortalized.     

F-18 FDG uptake of bone marrow on PET/CT scan: it’s correlation with CD38/CD138 expressing myeloma cells in bone marrow of patients with multiple myeloma

The percentage of myeloma cells in bone marrow is subsequently an important index of disease in patients with multiple myeloma (MM). Bone marrow myeloma cells can be detected by strong CD38/CD138 positivity and light scatter characteristics using flow cytometry. The aim of the study was to evaluate the relationship between the degree of F-18 fluorodeoxyglucose (F-18 FDG) uptake and the percentage of CD38/CD138 expressing myeloma cells in the bone marrow of patients with MM. A total of 31 patients with MM (14 females and 17 males, mean age 59.5 ± 1.9 years, range 29–80 years) were included in the study. All patients underwent FDG-positron emission tomography/computed tomography (PET/CT) scan within 2 weeks after the completion of the usual staging workup for MM, consisting of X-ray skeletal survey and hematological/biochemical parameters including complete blood count, liver and kidney function test, erythrocyte sedimentation rate, serum immunoglobulins, urine light chain excretion, C-reactive protein, β2-microglobulin, and bone marrow plasma cell infiltration. In all patients, flow cytometry was performed for assessing the percentage of CD38/CD138 expressing myeloma cells in the bone marrow samples. The extent of bone marrow FDG uptake on PET/CT scans was visually graduated using a qualitative scoring system as extension score (E-score) and also a semiquantitative scoring system defined as mean standardized uptake value (mSUV) of both femora. There was a statistically significant positive correlation between the percentage of CD38/CD138 expressing plasma cells in bone marrow and both mean qualitative (r = 0.616) and semiquantitative (r = 0.755) results of F-18 FDG uptakes. mSUV and E-score of bone marrow FDG uptake values were also correlated with serum beta-2-microglobulin levels (r = 0.523 and r = 479, respectively). mSUV of bone marrow FDG uptake values were also negatively correlated with serum albumin levels (r = −0.424), whereas there was no correlation between E-score and albumin levels. In conclusion, our results indicate that increased F-18 FDG uptake of bone marrow is related to the percentage of plasma cell infiltration of bone marrow in patients with MM. Therefore, F-18 FDG-PET/CT study may be a useful tool for predicting the levels of myeloma cells in bone marrow, and an additional analysis of FDG uptake of bone marrow on FDG-PET/CT scans should be performed in patients undergoing PET studies during the initial staging, evaluating the therapy response, and monitoring patients with MM.     

G-CSF therapy with mobilization of bone marrow stem cells for myocardial recovery after acute myocardial infarction-A relevant treatment?

This review of adjunctive therapy with subcutaneous granulocyte-colony stimulating factor (G-CSF) to patients with acute myocardial infarction (AMI) focus on the cardioprotective effects and potential mechanisms of G-CSF and discuss the therapeutic potential of G-CSF. All clinical trials published in peer-reviewed journals identified through PubMed are discussed. G-CSF treatment seems to be safe, and initial unblinded trials in patients with AMI were encouraging. However, larger double-blind placebo-controlled trials have not been able to demonstrate improved myocardial recovery after G-CSF treatment. Current controversies in interpretation of the results include 1) importance of direct cardiac effect of G-CSF vs indirect through bone marrow stem and progenitor cell mobilization, 2) importance of timing of G-CSF therapy, 3) importance of G-CSF dose, and 4) importance of cell types mobilized from the bone-marrow. Cell-based therapies to improve cardiac function remain promising and further experimental and clinical studies are warranted to determine the future role of G-CSF.     

A case of early gastric cancer with bone metastases: are bone marrow micrometastases significant?

Gastric adenocarcinoma is currently the 14th cause of death worldwide. Early gastric cancer, defined as cancer not penetrating deeper than the submucosa, is considered to carry an excellent prognosis with 5-year survival rates reaching more than 90%. Cases of bone metastases due to intramucosal gastric cancer are very rarely described. A case of a 70-year old male presenting with confirmed bone metastases 7 years after a curative resection for a mucosal gastric carcinoma is discussed. The patient was investigated with bone marrow biopsy and bone scan and showed no other signs of disease. The clinicopathologic features included poor differentiation, signet ring cells presence, no lymph node involvement and a negative second laparotomy two years after the initial surgery. Studies concerning the presence of residual disease in the form of bone marrow micrometastases are briefly reviewed emphasizing that intramucosal gastric cancer still carries the p sibility for metastasis, many years after a curative resection, mandating long term alertness from the attending physician.     

Adult bone marrow–derived cells: Regenerative potential, plasticity, and tissue commitment

Reconstitution of infarcted myocardium with functional new cardiomyocytes and vessels, a goal that only a few years ago would have been regarded as extravagant, is now actively pursued in numerous laboratories and clinical centers. Several recent studies in animals as well as humans have shown that transplantation of adult bone marrow-derived cells (BMCs) can improve left ventricular function and halt adverse remodeling after myocardial infarction. Differentiation of adult BMCs into cells of cardiac and vascular lineages has been proposed as a mechanism underlying these benefits and, indeed, differentiation of adult BMCs into cells of non-hematopoietic lineages, including cells of brain, skeletal muscle, heart, liver, and other organs, has been documented repeatedly both in vitro and in vivo. These results are in contrast with conventional definitions and dogma, according to which adult tissue-specific stem cells exhibit only restricted differentiation potential. Thus, these recent studies have sparked intense debate over the ability of adult BMCs to differentiate into non-hematopoietic tissues, and the regeneration of myocardium by differentiation of adult BMCs remains highly controversial. Because of the enormous clinical implications of BMC-mediated cardiac repair, numerous laboratories are currently addressing the feasibility of cardiac regeneration with BMCs and deciphering the mechanism underlying the beneficial effects. The purpose of this review is to critically examine the available evidence regarding the ability of adult BMCs to regenerate non-hematopoietic tissues and their utility in therapeutic cardiac regeneration.