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Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.

ABSTRACT: Bone marrow-derived stromal cells (BMSCs) are defined by their ability to self-renew and differentiate into at least three mesenchymal cell types (bone, adipose, and cartilage). The inability to isolate a reliably efficacious and homogeneous population of early progenitor cells has limited efforts to increase their therapeutic potential. In this study, we focused on identifying protein markers that may be employed to predict the efficacy of a cultured BMSC population. Markers of progenitor status were identified by comparing BMSCs at early and late passage, donor-matched skin fibroblasts, and commercially available dermal fibroblast cell lines. Differentiation potential was determined according to in vitro assays of osteogenesis, adipogenesis, and chondrogenesis. Early-passage BMSCs differentiated into all three lineages, whereas late-passage BMSCs and both fibroblast preparations did not. To identify novel markers of early progenitors, microarray transcript analysis between early-passage BMSCs and fibroblasts was performed. Messenger RNA encoding the cytokine leukemia inhibitory factor (LIF) was identified as differentially expressed. Enzyme-linked immunosorbent assay on conditioned media confirmed that LIF secretion was much higher from early progenitor BMSCs than donor-matched or commercial lines of fibroblasts and dropped with extensive expansion or induction of differentiation. In clonally expanded BMSCs, colonies that retained progenitor status expressed significantly higher levels of LIF than those that failed to differentiate. Our results indicate that LIF expression may represent a marker to quantify the differentiation potential of BMSCs and may be especially suited for the rapid, noninvasive quality control of clinical preparations.

SUBMITTER: Whitney MJ

PROVIDER: S-EPMC2809661 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.

Whitney Mandolin J MJ Lee Andy A Ylostalo Joni J Zeitouni Suzanne S Tucker Alan A Gregory Carl A CA

Tissue engineering. Part A 20090101 1

Bone marrow-derived stromal cells (BMSCs) are defined by their ability to self-renew and differentiate into at least three mesenchymal cell types (bone, adipose, and cartilage). The inability to isolate a reliably efficacious and homogeneous population of early progenitor cells has limited efforts to increase their therapeutic potential. In this study, we focused on identifying protein markers that may be employed to predict the efficacy of a cultured BMSC population. Markers of progenitor statu ...[more]

PMID: 18637760

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Project description:For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort-hazard ratio (95% confidence interval) of 2.50 (1-9.66); p = 0.05-together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

Project description:Hematopoietic stem cell transplantation (HSCT) is a common practice in the treatment of (malignant-) hematopoietic diseases. Umbilical Cord Blood (UCB) has become an important hematopoietic stem and progenitor cell (HSPC) source for allogeneic HSCT. However, the relatively low number of HSPCs that are present in a single UCB unit is associated with a delayed engraftment and a higher risk for graft failure. To overcome these limitations, the discovery of new therapeutics and the development of efficient ex vivo HSPC expansion conditions will help towards the successful treatment of malignant hematopoietic disease. Bone marrow derived mesenchymal stromal cells (BMSCs) are known to support the characteristic properties of HSPCs in the bone marrow microenvironment. Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support HSPC expansion, however, the mechanism and processes responsible for the cell-cell communication of EVs are still unknown. In the current study, we investigate whether primary human BMSC EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue respectively, can increase the relative low number of HSPC found in UCB and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs but not fEVs showed supportive ex vivo expansion capacity of UCB-HSPC. Taking advantage of the two BMSC sources with different supportive effects, we performed small RNA sequencing and proteomics analyses on the EV cargo and investigated how gene expression is modulated in HSPC after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the non-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. We identified well-defined EV markers such as ANXA1, ANXA2, ANXA5 and GAPDH as the most abundant proteins detected in both aEV and fEV. Gene Ontology (GO) analyses of the enriched proteins in aEVs and fEVs illustrated that of the proteins overrepresented in aEVs were annotated to oxidation-reduction process (eg. HADHA, HADHB), mitochondrial ATP synthesis coupled proton transport (eg. ATP5A1, ATP5B and ATP5O) or protein folding (eg. FKBP11, FKBP10, FKBP2, MESDC2). In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization (eg. FN1, COL12A1, COL1A1, COL6A1, CCDC80) or positive regulation of cell migration (eg. ITGA4, ITGA6, GDF15, SEMA3, APDSD6, MMP14, ICAM1, FGFR1, PDGFRA, ADAMTS1). Interestingly, we found various proteins that were overrepresented in the non-supportive fEV cargo, that are involved in transforming growth factor beta receptor (TGFBR) signaling pathway (TGFBR1, TGFBR2, TGFB1, TGFB2, LTBP1, LTBP2, BMPR1A, GDF5, GDF15, PARP1, RPS27A and COL3A1). Together this illustrates the large differences in the protein content of EV populations derived from two different origins. Small RNA sequencing identified different molecular signatures between the supportive aEVs and the non-supportive fEVs. miRNA, yRNA and piRNA were abundantly found in aEV, while fEVs contained a significant enrichment of snoRNAs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPC by targeting multiple proapoptotic genes, was highly and significantly enriched in aEV in comparison to fEV. Although we identified a significant difference in EV cargo and supportive effects of aEVs and fEVs, RNAseq analyses of 24hrs treated HSPCs with aEVs or fEVs indicated that only a limited set of genes was differentially regulated when compared to cells that were only treated with cytokines. Interestingly, we identified 5 genes, (eg MTF1, PER1, HOMER1, HSPA6, ENSG00000260534) that were significantly upregulated upon aEV compared to fEV treatment of HSPC. Moreover, HSPC treated with fEVs, resulted in increased expression of genes (SKIL, SMAD7, ENG, LDLRAD4) that are involved in the negative regulation of the TGFbeta signalling pathway. Together with the TGFbeta pathway proteins that were specifically identified in the fEVs, our results suggest that HSPCs may activate a negative feedback loop upon fEV treatment that consolidate HSPC expansion. In conclusion, our study gives novel insights into the complex biological role of EVs in the bone marrow microenvironment. Systematic analyses of supportive and non-supportive human BMSC derived extracellular vesicles indicated known molecules, eg. microRNA cluster miR-99b/let-7e/miR-125a, and the presence of TGFbeta pathway components that are important regulators in the cell-cell communication via EVs and open new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Dataset Information

Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.

Publications

Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.

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