Effects of JAK1/2 inhibition on bone marrow stromal cells of myeloproliferative neoplasm (MPN) patients and healthy individuals.

OBJECTIVE: Philadelphia-negative myeloproliferative neoplasms (MPNs) commonly share hyperactive JAK-STAT signaling affecting hematopoietic stem cells (HSC) and their progeny. The JAK1/2 inhibitor Ruxolitinib has remarkable clinical efficacy, including spleen reduction, improvement of constitutional symptoms, and bone marrow (BM) fibrosis reversal. Whether this is due to inhibition of JAK2-mutated HSC only, or whether Ruxolitinib also affects BM stroma is not known. METHODS: This study investigated potential effects of Ruxolitinib on BM mesenchymal stromal cells (MSC), which are not only major regulators of hematopoiesis but also contribute to fibrosis, from 10 healthy donors and 7 JAK2V617F -positive MPN patients. RESULTS: Ruxolitinib moderately inhibited the growth of healthy donor MSC (HD-MSC) and MSC from JAK2V617F+ MPN patients (P-MSC) in short- and long-term assays. The clonogenic potential of HD-MSC was not affected by Ruxolitinib. JAK-STAT signaling, however, was markedly inhibited in both HD-MSC and P-MSC, the latter of which showed higher expression of fibrosis-associated and hematopoiesis-maintenance genes. Moreover, Ruxolitinib reduced MSC secretion of MCP-1 and IL-6. CONCLUSION: Ruxolitinib affected JAK2 signaling in MSC at clinically relevant doses, which is likely to contribute to the normalization of the inflammatory milieu in MPNs. Thus, combined HSC and stroma-directed interventions have the potential to improve constitutional symptoms and reduce stromal proliferation in MPNs.

Investigating the effect of lead acetate on rat bone marrow-derived mesenchymal stem cells toxicity: role of apoptosis.

Lead exposure continues to be a significant public health problem. Osteoporosis, inhibition of fracture healing, and cartilage functional impairment have been reported from lead exposure. Mesenchymal stem cells (MSCs) are a bone marrow population of cells with the ability to differentiate into various cell types, particularly osteocytes and chondrocytes. Despite intensive investigation on the effect of lead poisoning on various cell types, there is very little if any report on the effect of lead on MSCs. The aim of this study, therefore, was to investigate the effect of lead acetate on rat bone marrow derived MSCs toxicity and its mechanism by examining the role of pro- and anti-apoptotic proteins in this process. It was revealed that lead acetate could induce cell death in a dose-dependent manner using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium (MTT) assay. Compared to controls, the significant over-expression of pro-apoptotic proteins, including Bax, caspases-9, -3, and p53, with no significant change in anti-apoptotic Bcl(2) protein were obtained in lead-treated cells using western blotting analysis. There was a significant increase in DNA fragmentation in treated MSCs compared to controls using flow-cytometry. Finally, it might be concluded that lead acetate could induce cell toxicity and apoptosis in MSCs, causing instability in mitochondria and in turn activation of the intrinsic pathway including over-expression of Bax, caspase 9 and caspase 3, leading to DNA damage and activation of P53.

In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).

Human Primary Bone Marrow Mesenchymal Stromal Cells and Their in vitro Progenies Display Distinct Transcriptional Profile Signatures.

Bone marrow mesenchymal stromal cells (BM-MSCs) are a rare population of cells that gives rise to skeletal tissues and the hematopoietic stroma in vivo. Recently, we have demonstrated that BM-MSCs fulfill stringent in vivo stem cell criteria when propagated as non-adherent mesenspheres but not as adherent-cultured cells. Motivated by these profound functional differences, the current study aimed to identify potential important MSC regulators by investigating global gene expression profiles of adherent and non-adherent culture-derived BM-MSCs in comparison with primary BM-MSCs. A substantial number of genes were differentially expressed between primary and culture-expanded cells already early upon culture, and numerous genes were found to be different when comparing adherent and non-adherent BM-MSCs. Cluster analysis identified 16 sets of genes of which two displayed comparable gene expression levels in primary and non-adherent cultured cells, but not in adherent cultured cells. This pattern suggested that these clusters contained candidate regulators of BM-MSCs. Gene expression differences were confirmed for selected genes and BM-MSC transcription factors by protein analysis and RT-PCR, respectively. Taken together, these data demonstrated profound gene expression changes upon culture of primary BM-MSCs. Moreover, gene cluster differences provide the basis to uncover the regulatory mechanisms that control primary and cultured BM-MSCs.

Isolation and characterization of primary bone marrow mesenchymal stromal cells.

Bone marrow (BM) contains a rare population of mesenchymal stromal cells (MSCs), which have been characterized as nonhematopoietic skeletal progenitor cells with central importance for the hematopoietic microenvironment. Classically, MSCs are isolated by plastic adherence and subsequent culture. However, as cultured stromal cells differ from their in vivo progenitors, it is important to identify the phenotype of the primary MSCs to study these cells in more detail. In the past years, several surface markers have been reported to be suitable for effective enrichment of BM-MSCs, and recent data indicate that the putative MSC stem/progenitor cell population in human adult BM is highly enriched in Lin(-) CD45(-) CD271(+) CD140a (PDGFRα)(low/-) cells. Moreover, surface marker combinations have been described for the isolation of MSCs from murine BM. On the basis of these findings, the role of primary MSCs can now be studied in normal and, importantly, diseased BM. Furthermore, genetically engineered mouse models have been developed as powerful tools to investigate well-defined BM stromal cell populations in vivo. Our discussion aims to provide a concise overview of the current state of the art in BM-MSC isolation in humans and briefly present murine MSC isolation approaches and genetic models.

Human Non-Hematopoietic CD271pos/CD140alow/neg Bone Marrow Stroma Cells Fulfill Stringent Stem Cell Criteria in Serial Transplantations.

Human bone marrow contains a population of non-hematopoietic stromal stem/progenitor cells (BMSCs), which play a central role for bone marrow stroma and the hematopoietic microenvironment. However, the precise characteristics and potential stem cell properties of defined BMSC populations have not yet been thoroughly investigated. Using standard adherent colony-forming unit fibroblast (CFU-F) assays, we have previously shown that BMSCs were highly enriched in the nonhematopoietic CD271pos/CD140alow/neg fraction of normal adult human bone marrow. In this study, we demonstrate that prospectively isolated CD271pos/CD140alow/neg BMSCs expressed high levels of hematopoiesis supporting genes and signature mesenchymal and multipotency genes on a single cell basis. Furthermore, CD271pos/CD140alow/neg BMSCs gave rise to non-adherent sphere colonies (mesenspheres) with typical surface marker profile and trilineage in vitro differentiation potential. Importantly, serial transplantations of CD271pos/CD140alow/neg BMSC-derived mesenspheres (single cell and bulk) into immunodeficient NOD scid gamma (NSG) mice showed increased mesensphere numbers and full differentiation potential after both primary and secondary transplantations. In contrast, BMSC self-renewal potential decreased under standard adherent culture conditions. These data therefore indicate that CD271pos/CD140alow/neg BMSCs represent a population of primary stem cells with MSC phenotype and sphere-forming capacity that fulfill stringent functional stem cell criteria in vivo in a serial transplantation setting.

Low/negative expression of PDGFR-α identifies the candidate primary mesenchymal stromal cells in adult human bone marrow.

Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key negative selection marker for adult human BM-MSCs, which enables to prospectively isolate a close to pure population of candidate human adult stroma stem/progenitor cells with potent hematopoiesis-supporting capacity.

Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion.

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets.

Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.