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Background Aims: Given their low immunogenicity, immunoregulatory effects and multiple differentiation capacity, mesenchymal stromal cells (MSCs) have the potential to be used for "off-the-shelf" cell therapy to treat various diseases. However, the allorejection of MSCs indicates that they are not fully immune-privileged. In this study, the authors investigated the immunogenicity of human adipose-derived MSCs (Ad-MSCs) and identified potential immunogenic molecules., Methods: To evaluate the immunogenicity of human Ad-MSCs in vivo, cells were transplanted into humanized mice (hu-mice), then T-cell infiltration and clearance of human Ad-MSCs were observed by immunofluorescence and bioluminescence imaging. One-way mixed lymphocyte reaction and flow cytometry were performed to evaluate the immunogenicity of human Ad-MSCs in vitro. High-throughput T-cell receptor (TCR) repertoire sequencing and mass spectrometry were applied to identified potential immunogenic molecules., Results: The authors observed that allogeneic Ad-MSCs recruited human T cells and caused faster clearance in hu-mice than non-humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The proliferation and activation of T cells were significantly enhanced during in vitro co-culture with human Ad-MSCs. In addition, the level of HLA-II expression on human Ad-MSCs was dramatically increased after co-culture with human peripheral blood mononuclear cells (PBMCs). High-throughput sequencing was applied to analyze the TCR repertoire of the Ad-MSC-recruited T cells to identify dominant TCR CDR3 sequences. Using synthesized TCR CDR3 peptides, the authors identified several potential immunogenic candidates, including alpha-enolase (ENO1). The ENO1 expression level of Ad-MSCs significantly increased after co-culture with PBMCs, whereas ENO1 inhibitor (ENOblock) treatment decreased the expression level of ENO1 and Ad-MSC-induced proliferation of T cells., Conclusions: The authors' findings improve the understanding of the immunogenicity of human Ad-MSCs and provide a theoretical basis for the safe clinical application of allogeneic MSC therapy., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Transcatheter arterial chemoembolization (TACE) and TACE in combination with sorafenib (TACE-sorafenib) have shown a significant survival benefit for the treatment of unresectable hepatocellular carcinoma (HCC). Adopting either as a first-line therapy carries major cost and resource implications. The objective of this study was to estimate the relative cost-effectiveness of TACE against TACE-sorafenib for unresectable HCC using a decision analytic model., Methods: A Markov cohort model was developed to compare TACE and TACE-sorafenib. Transition probabilities and utilities were obtained from systematic literature reviews, and costs were obtained from West China Hospital, Sichuan University, China. Survival benefits were reported in quality-adjusted life-years (QALYs). The incremental cost-effectiveness ratio (ICER) was calculated. Sensitive analysis was performed by varying potentially modifiable parameters of the model., Results: The base-case analysis showed that TACE cost $26 951 and yielded survival of 0.71 QALYs, and TACE-sorafenib cost $44 542 and yielded survival of 1.02 QALYs in the entire treatment. The ICER of TACE-sorafenib versus TACE was $56 745 per QALY gained, which was above threshold for cost-effectiveness in China. Sensitivity analysis revealed that the major driver of ICER was the cost post TACE-sorafenib therapy with stable state., Conclusion: TACE is a more cost-effective strategy than TACE-sorafenib for the treatment of unresectable HCC., (Copyright © 2017 The Editorial Board of Hepatobiliary & Pancreatic Diseases International. Published by Elsevier B.V. All rights reserved.)

Emerging evidence indicates that microRNAs (miRNA, or miR) play vital roles in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Elucidation of the molecular mechanisms that govern BMSCs osteogenic differentiation is of paramount importance for improving the treatment of bone-related diseases. In our current study, we investigated the role of miR-23a in BMSCs osteogenesis. Our results revealed that miR-23a was significantly downregulated during osteogenic differentiation. Overexpression of miR-23a inhibited osteogenic differentiation of hBMSCs in vitro, whereas downregulation of miR-23a enhanced the process. Target prediction analysis and dual luciferase reporter assays confirmed that low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5) was a direct target of miR-23a. Furthermore, knockdown of LRP5 inhibited osteogenic differentiation of hBMSCs, similar to the effect observed in upregulation miR-23a. Our data indicate that miR-23a plays an inhibitory role in osteogenic differentiation of hBMSCs, which may act by targeting LRP5., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Wound dressings of cell-laden bulk hydrogel or scaffold were mainly applied for enhanced cell engraftment in contrast to free cell injection. However, dressing of cells laden in biomaterials on wound surface might not effectively and timely exert functions on deep or chronic wounds where insufficient blood supply exists. Previously, we developed injectable gelatin microcryogels (GMs) which could load cells for enhanced cell delivery and cell therapy. In this study, biological changes of human adipose-derived stem cells (hASCs) laden in GMs were compared in varied aspects with traditional two dimensional (2D) cell culture, such as cell phenotype markers, stemness genes, differentiation, secretion of growth factors, cell apoptosis and cell memory by FACS, QRT-PCR and ELISA, that demonstrated the priming effects of GMs on upregulation of stemness genes and improved secretion of growth factors of hASCs for potential augmented wound healing. In a full-thickness skin wound model in nude mice, multisite injection and dressing of hASCs-laden GMs could significantly accelerate the healing compared to free cell injection. Bioluminescence imaging and protein analysis indicated improved cell retention and secretion of multiple growth factors. Our study suggests that GMs as primed injectable 3D micro-niches represent a new cell delivery methodology for skin wound healing which could not only benefit on the recovery of wound bed but also play direct effects on wound basal layer for healing enhancement. Injectable GMs as facile multisite cell delivery approach potentially provide new minimally-invasive therapeutic strategy for refractory wounds such as diabetic ulcer or radiative skin wound., Statement of Significance: This work applied a type of elastic micro-scaffold (GMs) to load and prime hMSCs for skin wound healing. Due to the injectability of GMs, the 3D cellular micro-niches could simply realize minimally-invasive and multisite cell delivery approach for accelerating the wound healing process superior to free cell injection. The biological features of MSCs has been thoroughly characterized during 3D culture in GMs (i.e. cell proliferation, characterization of cell surface markers, stemness of MSCs in GMs, differentiation of MSCs in GMs, secretion of MSCs in GMs, induced apoptosis of MSCs in GMs). Multiple methods such as bioluminescent imaging, immunohistochemistry, immunofluorescence, qRT-PCR, ELSA and western blot were used to assess the in vivo results between groups., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential, which makes them attractive targets for regenerative medicine applications. Efficient gene transfer into MSCs is essential for basic research in developmental biology and for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors (CARs), but not into MSCs, which lack CAR expression. To overcome this problem, an Adv coated with cationic polymer polyethyleneimine (PEI) was developed. In this study, we demonstrated that PEI coating with an optimal ratio can enhance adenoviral transduction of MSCs without cytotoxicity. We also investigated the physicochemical properties and internalization mechanisms of the PEI-coated Adv. These results could help to evaluate the potentiality of the PEI-coated Adv as a prototype vector for efficient and safe transduction into MSCs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Glutamate excitotoxicity has been implicated as one of the factors contributing to neuronal apoptosis and is involved in many neurodegenerative diseases. Previous studies suggest that mesenchymal stem cells have the ability to protect cultured neurons from excitotoxicity-induced apoptosis, although the underlying mechanisms are not clear. In this study, we evaluated whether adipose mesenchymal stem cells (AMSCs) could protect against glutamate-induced injury in PC12 cells by secreting neurotrophic factors. We found that AMSCs secreted neurotrophic factors including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) under both normoxic and hypoxic conditions. AMSC - conditioned medium (AMSC-CM) had a protective effect on excitotoxicity-injured PC12 cells, as indicated by increased cell viability, decreased number of TUNEL-staining positive nuclei and lowered caspase-3 activity. By using neutralizing monoclonal antibodies and specific inhibitors, VEGF, HGF and BDNF were identified as the mediators of AMSC effects and PI3-K/Akt and MAPK pathways were involved. Western blot analysis showed that AMSC-CM can increase the level of p-Akt, up-regulate XIAP and reduce the level of cleaved-caspase-3 in PC12 cells. These results suggest that AMSCs can effectively protect PC12 cells from glutamate excitotoxicity-induced apoptosis and support the hypothesis that AMSCs may be a useful treatment for stroke or neurodegenerative diseases which often involve excitotoxicity., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

On the basis of the recent report that granulocyte colony-stimulating factor (G-CSF) administration after rats' partial orthotopic liver transplantation greatly improved survival rate and liver regeneration of partial graft, we here evaluated the effect of intermittent administration of G-CSF on fibrosis formation induced by carbon tetrachloride (CCl(4)). Bone marrow chimeric female C57BL/6 mice were treated with G-CSF at days 1, 7, 14, 21, and 28 after CCl(4) challenge. At day 35 after CCl(4) administration, we found that G-CSF treatment significantly reduced CCl(4)-induced liver damage and collagen deposition. In addition, levels of hepatic hydroxyproline and serum fibrosis markers in mice receiving G-CSF administration after CCl(4) challenge were significantly lower compared to those of control mice. Histological examination suggested that hepatic damage recovery was much better in these G-CSF-treated mice. Immunofluorescence and fluorescence in situ hybridization (FISH) analysis revealed that donor cells engrafted into host liver, had epithelium-like morphology and expressed albumin, although at low frequency. These results suggest that intermittent G-CSF treatment might initiate endogenous hepatic tissue regeneration in response to CCl(4) injury and ameliorate its fibrogenic effects., ((c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Human bone marrow mesenchymal stem cells (MSCs) are important cell population located in bone marrow that are thought to have multiple functions in cell transplantation and gene therapy. Although in vitro experiments have demonstrated that hMSCs are resistant to apoptosis induction by DNA damage agents such as chemotherapeutic substances used in bone marrow transplantation, the molecular mechanism underlying remains unclear. p73 is highly similar to p53 and plays crucial roles in regulating DNA damage-induced apoptosis pathways. In this study, we investigated the role of p73 alpha in response to chemotherapeutic substances in cultured human bone marrow MSCs. Cellular chemosensitivity and DNA damage-induced apoptotic cell death were examined in the hMSCs with exogenously over-expressed p73 alpha. Our results showed that the expression of retrovirus-driven human p73 alpha could be successfully induced in hMSCs, the over-expression of ectopic p73 alpha resulted in a significant increase of cellular sensitivity to cisplatin. The increase of cellular apoptosis was attributed to enhanced chemosensitivity in p73 alpha infected cells. Moreover, immunoblot analysis indicated that the co-activation of pro-apoptotic factors Bax and p21 were observed in the p73 alpha infected cells after cisplatin treatment. In conclusion, our findings suggested that p73 alpha is an important determinant of cellular chemosensitivity in human bone marrow MSCs., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, exert immunomodulatory effects on immune cells, even dendritic cells (DCs). However, whether they influence the destiny of full mature DCs (maDCs) remains controversial. Here we report that MSCs vigorously promote proliferation of maDCs, significantly reduce their expression of Ia, CD11c, CD80, CD86, and CD40 while increasing CD11b expression. Interestingly, though these phenotypes clearly suggest their skew to immature status, bacterial lipopolysaccharide (LPS) stimulation could not reverse this trend. Moreover, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-treated maDCs (MSC-DCs) were also observed. Furthermore we found that MSCs, partly via cell-cell contact, drive maDCs to differentiate into a novel Jagged-2-dependent regulatory DC population and escape their apoptotic fate. These results further support the role of MSCs in preventing rejection in organ transplantation and treatment of autoimmune disease.

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.

The hypoxia-inducible factor (HIF) 1alpha is a key regulator of the cellular response to oxygen deprivation. Specific disruption of the HIF-1 pathway is important for exploring its role in tumor biology and developing more efficient weapons to treat cancer. In this study, we stably transfected human breast tumor MCF-7 cells with short hairpin RNA expression vectors targeting HIF-1alpha. After knockdown of HIF-1alpha, hypoxia-induced expression of its target genes such as vascular endothelial growth factor, Glut-1, phosphoglycerate kinase, and P-glycoprotein were markedly attenuated. Moreover, HIF-1alpha knockdown was found to suppress the shift from S-phase to G(1) induced by hypoxia and increase drug sensitivity to methotrexate. The growth rates of HIF1alpha-knockdown tumors were drastically retarded in both subcutaneous and orthotopic xenograft models, which were accompanied by decreased angiogenesis and reduced expression of glucose transporter in tissue sections. These data demonstrate that HIF-1alpha knockdown reduces tumorigenicity of MCF-7 cells and suggest a promising combination of both anti-HIF-1 strategy and traditional chemotherapy to improve cancer treatment.

In mucopolysaccharidosis-I (MPS-I), alpha-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2-FGFR1-HS interactions, resulting in defective FGF-2-induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS-cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

In this study, we isolated CD31(-), CD34(-), CD106(-) (VCAM-1(-)), and fetal liver kinase(+) (Flk1(+)) cells from adipose tissue. These cells can be induced to differentiate into cells of osteogenic and adipogenic lineages in vitro and were termed adipose derived adult stem cells (ADAS cells). We also showed that they have characteristics of endothelial progenitor cells. In vitro, ADAS cells expressed endothelial markers when cultured with VEGF. In vivo, ADAS cells can differentiate in response to local cues into endothelial cells that contributed to neoangiogenesis in hindlimb ischemia models. PI3 kinase inhibitor LY294002 blocked the differentiation of ADAS cells into endothelial cells in vitro. Because ADAS cells can be expanded in culture without obvious senescence for more than 20 population doublings, they may be a potential source of endothelial cells for cellular pro-angiogenic therapies.

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly, the BCR/ABL fusion gene, which is present in chronic myelogenous leukemia (CML), was also detected in the endothelial cells of patients with CML, suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells, thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells.

Mesenchymal stem cells (MSCs) are an important cell population in the bone-marrow microenvironment and are considered to be engaged mainly in the support of hematopoiesis. Recent work has shown that MSCs also have profound immunomodulatory function, both in vitro and in vivo. Because MSCs can be expanded rapidly to the numbers required for clinical application, several preclinical and clinical studies have been performed in the areas of immune diseases and bone-marrow transplantation. In this review we discuss the mechanisms underlying the MSC's immunomodulating properties and its potential applications.

We report that a cell population derived from human fetal bone marrow, termed Flk1+CD34- multipotent stem cells, can differentiate not only into osteogenic, adipogenic, and endothelial lineages but also into hepatocyte-like cells and neural and erythroid cells at the single-cell level. We depleted mononuclear cells from fetal bone marrow of CD45+, GlyA+, and CD34+ cells with the use of micromagnetic beads, then cultured them by limiting dilution. Three single colonies were harvested, expanded, and characterized. The clones have been expanded for more than 50 cell doublings, and cell-doubling time was about 30 hours. About 90% cells were in the G(0)/G(1) phase of the cell cycle, and the cells from the single colony maintained Flk1+ and CD34-. Because fetal bone marrow-derived Flk1+CD34-multipotent stem cells have the capacity for self-renewal and multilineage differentiation even after being expanded for more than 50 cell doublings, they may be an ideal source of stem cells for the treatment of inherited or degenerative diseases.

Mesenchymal stem cells (MSCs) have been cultured from many sources, including bone marrow and liver. To further support our hypothesis that MSCs exist in most postnatal tissues, we isolated a clonogenic, multipotent, rapidly proliferating population of cells from a fetal pancreas and termed them "pancreas-derived mesenchymal stem cells" (PMSCs). They withstood being passaged as many as 30 times without sustaining significant structural changes. In this study, we showed that PMSCs are positive for CD44, CD29, and CDI3 but negative for CD34 and HLA-DR and that they stained with collagen I and III but not with von Willebrand factor antibody. During the log phase of growth, PMSCs proliferated, doubling in population in about 30 hours. Cell-cycle analysis showed that more than 90% of cells were in the G0 and G1 phases, whereas a small subpopulation of cells were actively engaged in proliferation (S + G2 + M = 3.55%). Under differentiation culture conditions, PMSCs differentiated into cells of osteogenic, chondrogenic, and adipogenic lineages. These results demonstrate that PMSCs can be isolated from human fetal pancreas by means of their adherent ability and that they are capable of self-renewal, propagation, and multipotent differentiation.

Most insights into the molecular mechanisms underlying transformation by the p210(BCR/ABL) oncoprotein are derived from studies in which BCR/ABL cDNA was introduced into hematopoietic or fibroblast cell lines. However, such cell line models may not represent all the features of chronic myelogenous leukemia (CML) caused by additional genetic abnormalities and differences in the biology of cell lines compared with primary hematopoietic progenitor and stem cells. A primary human hematopoietic progenitor cell model for CML was developed by the transduction of b3a2 BCR/ABL cDNA in normal CD34(+) cells. Adhesion of BCR/ABL-transduced CD34(+) cells to fibronectin was decreased, but migration over fibronectin was enhanced compared with that of mock-transduced CD34(+) cells. Adhesion to fibronectin did not decrease the proliferation of BCR/ABL-transduced CD34(+) cells but decreased the proliferation of mock-transduced CD34(+) cells. This was associated with elevated levels of p27(Kip) in p210(BCR/ABL)-expressing CD34(+) cells. In addition, the presence of p210(BCR/ABL) delayed apoptosis after the withdrawal of cytokines and serum. Finally, significantly more and larger myeloid colony-forming units grew from BCR/ABL than from mock-transduced CD34(+) cells. Thus, the transduction of CD34(+) cells with the b3a2-BCR/ABL cDNA recreates most, if not all, phenotypic abnormalities seen in primary CML CD34(+) cells. This model should prove useful for the study of molecular mechanisms associated with the presence of p210(BCR/ABL) in CML.

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ABL gene rearrangement. The only curative therapy is allogeneic transplantation. Although autologous transplants may prolong survival, most patients relapse because of disease persisting in the host and in the graft. Continued administration of chemotherapy after transplant could reduce the incidence of relapse provided that the autograft can be protected by transfer of a drug-resistance gene. However, CML autografts will almost certainly contain malignant stem cells that will also be rendered drug-resistant. The presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CML. We thus hypothesized that transfer of a vector that combines a drug-resistance gene with anti-BCR/ABL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX)-resistant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS sequences directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and MO7e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210(BCR/ABL) protein levels by 6- to 10-fold in most cells. This subsequently led to the restoration of normal function of BCR/ABL cDNA+ cells: they grew significantly slower in the presence of interleukin-3 (IL-3); they underwent apoptotic cell death when cultured without IL-3; and they had restored expression and function of adhesion receptors. These effects were specific, because LasBD-containing AS sequences directed at the b3a2 BCR/ABL breakpoint did not affect p190(BCR/ABL)-containing cells. LasBD also rendered 20% to 30% of primary Ph- and Ph+ CD34(+) cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resistant Ph+ CD34(+) cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitro and for more than 70 days in vivo. Finally, LasBD decreased tumorigenicity of 32DBCR/ABL cells in vivo by 3 to 4 logs. In conclusion, the tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD can suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prove to be an extremely useful adjunct in autologous transplantation for CML.

Hematopoiesis takes place in close contact with the marrow microenvironment. Normal progenitors adhere through a variety of receptors to stroma and extracellular matrix components, including fibronectin. Adhesion through beta1-integrin receptors to fibronectin not only anchor progenitors to the stroma but also result in direct adhesion-mediated signaling that inhibits progenitor proliferation. In contrast to normal hematopoiesis, chronic myelogenous leukemia (CML) is characterized not only by abnormal, premature circulation of primitive progenitors in the blood but also by continuous progenitor proliferation. Although CML progenitors express the same integrin receptors as normal progenitors, they fail to adhere to stroma and fibronectin, suggesting structural or functional abnormalities of these receptors. Furthermore, CML cells present in contact with stroma or fibronectin continue to proliferate, suggesting that failure to adhere through integrin receptors may also underlie the abnormal proliferation of CML progenitors. The observation that integrin-mediated adhesion and proliferation-inhibitory signaling can be restored through treatment with interferon-alpha or an activating anti-beta1-integrin antibody suggests a functional rather than structural defect that may be related to the presence of the BCR/ABL gene rearrangement in these cells. Insights into the role of integrins as adhesion molecules but also receptors that instruct hematopoietic progenitors to survive, proliferate, and possibly differentiate will not only further our understanding of the normal hematopoietic process but also provide insights into diseases characterized by deranged adhesion and proliferation that may lead to novel therapeutic approaches.