Karlien Pieters

A rapid, simple, and reproducible method for the isolation of mesenchymal stromal cells from wharton's jelly without enzymatic treatment

The co-infusion of mesenchymal stromal cells (MSCs) with hematopoietic stem cells could improve the hematopoietic engraftment after cord blood transplant. Adult bone marrow is the major source of MSCs for cell therapy. However, bone marrow aspiration involves an invasive procedure and, in the case of a cord blood transplant, requires the use of a third party. The umbilical cord matrix, called Whartons jelly (WJ), was previously shown to be a valuable source of MSCs. However, the process of cell separation is not standardized and needs to be optimized. In this study, we focused on the efficiency of the isolation procedure and expansion of cells from WJ MSCs isolated from human full-term umbilical cords. MSCs were isolated from the WJ without enzyme digestion or dissection. The procedure was based only on the plastic adhesion capacities of MSCs. Briefly, umbilical cord segments of 5-10 cm were cut longitudinally and plated with the WJ onto a plastic surface for 5 days in an appropriate culture medium. After removing the cord segment, the culture was pursued until subconfluency. The number of cells and their phenotypes, clonogenic capacities, differentiation capacities, immunomodulation, and hematopoietic supportive functions were evaluated. Using this method, we were able to isolate MSCs from all human umbilical cords analyzed (n = 50). We obtained a mean of 1.4 × 10(8) cells at the second passage and >7 × 10(9) cells at the third. The expanded cells expressed characteristic markers and presented typical functional properties of MSCs such as differentiation capacities, immunologic properties, and hematopoietic supportive functions. In conclusion, we have established a simple, rapid, and reproducible protocol to isolate abundant MSCs from short segments of umbilical cords.

Evidences of Early Senescence in Multiple Myeloma Bone Marrow Mesenchymal Stromal Cells

Background In multiple myeloma, bone marrow mesenchymal stromal cells support myeloma cell growth. Previous studies have suggested that direct and indirect interactions between malignant cells and bone marrow mesenchymal stromal cells result in constitutive abnormalities in the bone marrow mesenchymal stromal cells. Design and Methods The aims of this study were to investigate the constitutive abnormalities in myeloma bone marrow mesenchymal stromal cells and to evaluate the impact of new treatments. Results We demonstrated that myeloma bone marrow mesenchymal stromal cells have an increased expression of senescence-associated β-galactosidase, increased cell size, reduced proliferation capacity and characteristic expression of senescence-associated secretory profile members. We also observed a reduction in osteoblastogenic capacity and immunomodulatory activity and an increase in hematopoietic support capacity. Finally, we determined that current treatments were able to partially reduce some abnormalities in secreted factors, proliferation and osteoblastogenesis. Conclusions We showed that myeloma bone marrow mesenchymal stromal cells have an early senescent profile with profound alterations in their characteristics. This senescent state most likely participates in disease progression and relapse by altering the tumor microenvironment.

AMD3100 disrupts the cross-talk between chronic lymphocytic leukemia cells and a mesenchymal stromal or nurse-like cell-based microenvironment: pre-clinical evidence for its association with chronic lymphocytic leukemia treatments.

Background Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface. Design and Methods Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model. Results AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies. Conclusions Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.

Isolation of Adipose-Derived Stromal Cells Without Enzymatic Treatment: Expansion, Phenotypical, and Functional Characterization

Stem cell therapy is a potential method for the treatment of numerous diseases. The most frequent cellular source is bone-marrow-derived mesenchymal stromal cells (BM-MSCs). Human adipose-derived stromal cells (ADSCs) share similar properties with BM-MSCs as they support hematopoiesis, modulate ongoing immune responses, and differentiate into cells of mesodermal origin. On the other hand, ADSCs have higher frequency in situ, higher availability, and very few ethical issues compared with BM-MSCs, giving them an advantage over BM-MSCs for clinical use. Most of the methods used to isolate ADSCs contain a collagenase digestion step, but the type of collagenase and time of sample digestion vary among studies and these differences could have an impact on the cell properties and thus in result comparison. To overcome this obstacle, we propose a new method to isolate ADSCs from lipoaspirate without collagenase digestion step. We compared ADSCs obtained with our method versus classical protocol using collagenase digestion. Cells obtained with our method are equivalent but they have a better long-term hematopoietic support than those obtained with classical method. Moreover, our method has an advantage over the classical one as it is easier, safer, faster, less expensive, and more consistent with good manufacturing practices to obtain large number of ADSCs ex vivo.

A molecular score by quantitative PCR as a new prognostic tool at diagnosis for chronic lymphocytic leukemia patients.

Background Several markers have been proposed to predict the outcome of chronic lymphocytic leukemia (CLL) patients. However, discordances exist between the current prognostic factors, indicating that none of these factors are totally perfect. Methodology/Principal Findings Here, we compared the prognostic power of new RNA-based markers in order to construct a quantitative PCR (qPCR) score composed of the most powerful factors. ZAP70, LPL, CLLU1, microRNA-29c and microRNA-223 were measured by real time PCR in a cohort of 170 patients with a median follow-up of 64 months (range3-330). For each patient, cells were obtained at diagnosis and RNA was extracted from purified CD19 cells. The best markers were included in a qPCR score, which was thereafter compared to each individual factor. Statistical analysis showed that all five RNA-based markers can predict treatment-free survival (TFS), but only ZAP70, LPL and microRNA-29c could significantly predict overall survival (OS). These three markers were thus included in a simple qPCR score that was able to significantly predict TFS and OS by dividing patients into three groups (0/3, 1-2/3 and 3/3). Median TFS were >210, 61 and 24 months (P<0.0001) and median OS were >330, 242 and 137 months (P<0.0001), respectively. Interestingly, TFS results were also confirmed in Binet stage A patients (P<0.0001). When compared to other classical factors, this score displays the highest univariate Cox hazard ratio (TFS: HR = 9.45 and OS: HR = 13.88) but also provides additional prognostic information. Conclusions In our hands, this score is the most powerful tool for CLL risk stratification at the time of diagnosis.

Mesenchymal stromal cells from the foreskin: Tissue isolation, cell characterization and immunobiological properties

BACKGROUND AIMS Because of their self-renewal capacity, multilineage potential and immunomodulatory properties, MSCs are an attractive tool for cell-based immunotherapy strategies. Foreskin, considered as a biological waste material, has been shown to be a reservoir of therapeutic cells. METHODS MSCs were isolated from different foreskin samples, maintained under in vitro culture and defined according to the International Society for Cellular Therapy (ISCT) criteria. We subsequently determined their main cell characteristics as well as their immunobiological properties. The following parameters were determined: (i) morphology and phenotype, (ii) proliferative and clonogenic potentials, (iii) tri-lineage differentiation ability, (iv) immunological profile, (v) immunomodulatory properties and (vi) protein and messenger RNA expression/secretion profile of immunoregulatory cytokines/factors as well as the pattern of toll-like receptors (TLRs). By using a pro-inflammatory cytokine cocktail, we also evaluated the influence of an inflammatory environment on their biology. RESULTS With a typical fibroblast-like morphology and an ISCT-compliant phenotype, foreskin-MSCs (FSK-MSCs) were highly proliferative and had a great clonogenic potential. They displayed multilineage capacities and interesting immunomodulatory properties. Of importance, FSK-MSCs were not immunogenetic and were further able to inhibit T-cell proliferation. We showed that several immunoregulatory cytokines and factors might be potentially involved in FSK-MSC immunomodulation with particular attention to hepatocyte growth factor and interleukin-11. Moreover, FSK-MSCs expressed several TLRs and were sensitive to the inflammatory environment by properly adjusting their profile and fate. CONCLUSIONS Foreskin represents a new alternative source for MSCs that is compliant with ISCT criteria. Their unique immunobiological properties allow consideration of FSK-MSCs as a valuable tolerogenic product for cell-based immunotherapy.

Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control

Background Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. Materials and Methods We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Results Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). Conclusion We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

In vivo production of mesenchymal stromal cells after injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor in the bone marrow of healthy volunteers.

Autologous mesenchymal stromal cell (MSC)-based therapies offer one of the most promising and safe methods for regeneration or reconstruction of tissues and organs. Routine procedures to obtain adequate amount of autologous stem cells need their expansion through culture, with risks of contamination and cell differentiation, leading to the loss of cell ability for therapies. We suggest the use of human bone marrow (BM) as a physiological bioreactor to produce autologous MSC by injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor (rhsTF) in iliac crest. A trial on 13 healthy volunteers showed the feasibility and harmlessness of the procedure. The phenotype and cellularity of BM cells were not modified, on day 3 after injection. Endothelial progenitor cells (EPC) were mobilized to the bloodstream, without stimulation of hematopoietic stem cells (HSC). MSC level in BM increased with a specific commitment to preosteoblastic cell population both in vivo and in vitro. This self-stimulation system of BM seems thus to be a promising feasible process 3 days before clinical cell therapy applications.

Extracellular vesicles of bone marrow stromal cells rescue chronic lymphocytic leukemia B-cells from apoptosis, enhance their migration and induce gene expression modifications

Interactions between chronic lymphocytic leukemia (CLL) B cells and the bone marrow (BM) microenvironment play a major function in the physiopathology of CLL. Extracellular vesicles (EVs), which are composed of exosomes and microparticles, play an important role in cell communication. However, little is known about their role in CLL / microenvironment interactions. In the present study, EVs purified by ultracentrifugation from BM mesenchymal stromal cell (BM-MSC) cultures were added to CLL B cells. After their integration into CLL B cells, we observed a decrease of leukemic cell spontaneous apoptosis and an increase in their chemoresistance to several drugs, including fludarabine, ibrutinib, idelalisib and venetoclax after 24 hours. Spontaneous (P=0.0078) and stromal cell-derived factor 1α -induced migration capacities of CLL B cells were also enhanced (P=0.0020). A microarray study highlighted 805 differentially expressed genes between leukemic cells cultured with or without EVs. Of these, genes involved in the B-cell receptor pathway such as CCL3/4, EGR1/2/3, and MYC were increased. Interestingly, this signature presents important overlaps with other microenvironment stimuli such as B-cell receptor stimulation, CLL/nurse-like cells co-culture or those provided by a lymph node microenvironment. Finally, we showed that EVs from MSCs of leukemic patients also rescue leukemic cells from spontaneous or drug-induced apoptosis. However, they induce a higher migration and also a stronger gene modification compared to EVs of healthy MSCs. In conclusion, we show that EVs play a crucial role in CLL B cells/BM microenvironment communication.

The light chain IgLV3-21 defines a new poor prognostic subgroup in Chronic Lymphocytic Leukemia: results of a multicenter study

Purpose: Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21. Experimental Design: We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing. Results: Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (P < 0.0001) as well as MYC target genes (P = 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (P < 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements—similar to UM IgHV status—conferred poor prognosis compared with mutated IgHV (P < 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (P < 0.0001). Conclusions: We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis. Clin Cancer Res; 24(20); 5048–57. ©2018 AACR.