Electromagnetic Fields Generated by the IteraCoil Device Differentiate Mesenchymal Stem Progenitor Cells Into the Osteogenic Lineage.

Rapid advances in mesenchymal stem progenitor cells (MSPCs) have rendered impetus into the area of cell therapy and regenerative medicine. The main promise of future stem cell therapies is their reliance on autologous stem cells derived from adipose tissue, which also includes treatments of bone fractures and degeneration. The effectiveness of different electric devices utilized to reprogram MSPCs toward osteogenic differentiation has provided varying degrees of effectiveness for clinical use. Adipose tissue-derived MSPCs were flow-cytometrically characterized and further differentiated into osteoblasts by culturing either in growth medium with pro-osteogenic supplements or without supplements with alternating electromagnetic field (EMF) generated by IteraCoil. IteraCoil is a multi-solenoid coil with a specific complex geometry that creates a 3D-EMF with desired parameters without directly applying electrodes to the cells and tissues. The flow-cytometric analysis of highly enriched (≥95%) adipose-derived MSPCs (CD34- , CD73+ , CD90+ , and CD105+ ) was utilized for the study. Osteoblasts and chondrocyte differentiations were then assessed by specific staining and quantified using ImageJ (National Institutes of Health). The osteoblastic differentiation of MSPCs cultured in regular medium and exposed to EMF at 0.05 and 1 kHz frequencies was compared with MSPCs cultured in a pro-osteogenic supplemented medium. In this study, we demonstrated that EMF from IteraCoil might have affected the signaling pathways that induce the osteogenic differentiation of human adipose-derived MSPCs in the absence of exogenous osteogenic factors. Therefore, EMF-generated osteogenic differentiation of reprogrammed adipose-derived autologous MSPCs may treat the loss of osteoblasts and osteoporosis and open new avenues for the development of regenerative cellular therapy. © 2022 Bioelectromagnetics Society.

Epigenetic reprogramming induces the expansion of cord blood stem cells.

Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune-deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts.

Coactivator MYST1 regulates nuclear factor-κB and androgen receptor functions during proliferation of prostate cancer cells.

In prostate cancer (PCa), the functional synergy between androgen receptor (AR) and nuclear factor-κ B (NF-κB) escalates the resistance to therapeutic regimens and promotes aggressive tumor growth. Although the underlying mechanisms are less clear, gene regulatory abilities of coactivators can bridge the transcription functions of AR and NF-κB. The present study shows that MYST1 (MOZ, YBF2 and SAS2, and TIP60 protein 1) costimulates AR and NF-κB functions in PCa cells. We demonstrate that activation of NF-κB promotes deacetylation of MYST1 by sirtuin 1. Further, the mutually exclusive interactions of MYST1 with sirtuin 1 vs AR regulate the acetylation of lysine 16 on histone H4. Notably, in AR-lacking PC3 cells and in AR-depleted LNCaP cells, diminution of MYST1 activates the cleavage of poly(ADP-ribose) polymerase and caspase 3 that leads to apoptosis. In contrast, in AR-transformed PC3 cells (PC3-AR), depletion of MYST1 induces cyclin-dependent kinase (CDK) N1A/p21, which results in G2M arrest. Concomitantly, the levels of phospho-retinoblastoma, E2F1, CDK4, and CDK6 are reduced. Finally, the expression of tumor protein D52 (TPD52) was unequivocally affected in PC3, PC3-AR, and LNCaP cells. Taken together, the results of this study reveal that the functional interactions of MYST1 with AR and NF-κB are critical for PCa progression.

Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells.

Presently, blood transfusion products (TPs) are composed of terminally differentiated cells with a finite life span. We have developed an ex vivo-generated TP composed of erythroid progenitor cells (EPCs) and precursors cells. Several histone deacetylase inhibitors (HDACIs) were used in vitro to promote the preferential differentiation of cord blood (CB) CD34(+) cells to EPCs. A combination of cytokines and valproic acid (VPA): (1) promoted the greatest degree of EPC expansion, (2) led to the generation of EPCs which were capable of differentiating into the various stages of erythroid development, (3) led to epigenetic modifications (increased H3 acetylation) of promoters for erythroid-specific genes, which resulted in the acquisition of a gene expression pattern characteristic of primitive erythroid cells, and (4) promoted the generation of a TP that when infused into NOD/SCID mice produced mature RBCs containing both human adult and fetal globins as well Rh blood group Ag which persisted for 3 weeks and the retention of human EPCs and erythroid precursor cells within the BM of recipient mice. This ex vivo-generated EPC-TP likely represents a paradigm shift in transfusion medicine because of its potential to continue to generate additional RBCs after its infusion.

Epigenetic abnormalities in myeloproliferative neoplasms: a target for novel therapeutic strategies.

The myeloproliferative neoplasms (MPNs) are a group of clonal hematological malignancies characterized by a hypercellular bone marrow and a tendency to develop thrombotic complications and to evolve to myelofibrosis and acute leukemia. Unlike chronic myelogenous leukemia, where a single disease-initiating genetic event has been identified, a more complicated series of genetic mutations appear to be responsible for the BCR-ABL1-negative MPNs which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Recent studies have revealed a number of epigenetic alterations that also likely contribute to disease pathogenesis and determine clinical outcome. Increasing evidence indicates that alterations in DNA methylation, histone modification, and microRNA expression patterns can collectively influence gene expression and potentially contribute to MPN pathogenesis. Examples include mutations in genes encoding proteins that modify chromatin structure (EZH2, ASXL1, IDH1/2, JAK2V617F, and IKZF1) as well as epigenetic modification of genes critical for cell proliferation and survival (suppressors of cytokine signaling, polycythemia rubra vera-1, CXC chemokine receptor 4, and histone deacetylase (HDAC)). These epigenetic lesions serve as novel targets for experimental therapeutic interventions. Clinical trials are currently underway evaluating HDAC inhibitors and DNA methyltransferase inhibitors for the treatment of patients with MPNs.

Computer aided identification of small molecules disrupting uPAR/alpha5beta1--integrin interaction: a new paradigm for metastasis prevention.

BACKGROUND: Disseminated dormant cancer cells can resume growth and eventually form overt metastases, but the underlying molecular mechanism responsible for this change remains obscure. We previously established that cell surface interaction between urokinase receptor (uPAR) and alpha5beta1-integrin initiates a sequel of events, involving MAPK-ERK activation that culminates in progressive cancer growth. We also identified the site on uPAR that binds alpha5beta1-integrin. Disruption of uPAR/integrin interaction blocks ERK activation and forces cancer cells into dormancy. METHODS AND PRINCIPLE FINDINGS: Using a target structure guided computation docking we identified 68 compounds from a diversity library of 13,000 small molecules that were predicted to interact with a previously identified integrin-binding site on uPAR. Of these 68 chemical hits, ten inhibited ERK activation in a cellular assay and of those, 2 compounds, 2-(Pyridin-2-ylamino)-quinolin-8-ol and, 2,2'-(methylimino)di (8-quinolinol) inhibited ERK activation by disrupting the uPAR/integrins interaction. These two compounds, when applied in vivo, inhibited ERK activity and tumor growth and blocked metastases of a model head and neck carcinoma. CONCLUSIONS/SIGNIFICANCE: We showed that interaction between two large proteins (uPAR and alpha5beta1-integrin) can be disrupted by a small molecule leading to profound downstream effects. Because this interaction occurs in cells with high uPAR expression, a property almost exclusive to cancer cells, we expect a new therapy based on these lead compounds to be cancer cell specific and minimally toxic. This treatment, rather than killing disseminated metastatic cells, should induce a protracted state of dormancy and prevent overt metastases.

A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth.

Highly expressed urokinase plasminogen activator receptor (uPAR) can interact with alpha5beta1 integrin leading to persistent ERK activation and tumorigenicity. Disrupting this interaction reduces ERK activity, forcing cancer cells into dormancy. We identified a site in uPAR domain III that is indispensable for these effects. A 9-mer peptide derived from a sequence in domain III (residues 240-248) binds purified alpha5beta1 integrin. Substituting a single amino acid (S245A) in this peptide, or in full-length soluble uPAR, impairs binding of the purified integrin. In the recently solved crystal structure of uPAR the Ser-245 is confined to the large external surface of the receptor, a location that is well separated from the central urokinase plasminogen binding cavity. The impact of this site on alpha5beta1 integrin-dependent cell functions was examined by comparing cells induced to express uPAR(wt) or the uPAR(S245A) mutant. Transfecting uPAR(wt) into cells with low endogenous levels of uPAR, inactive integrin, low ERK activity, and a dormant phenotype in vivo restores these functions and reinstates growth in vivo. In contrast, transfection of the same cells with uPAR(S245A) elicits only very small changes. Incubation of highly malignant cells with the wild-type, but not the S245A mutant peptide, disrupts the uPAR integrin interaction leading to down-regulation of ERK activity. The relevance of this binding site, and of the lateral uPAR-alpha5beta1 integrin interaction, to ERK pathway activation and tumor growth implicates it as a possible specific target for cancer therapy.

ERK couples chronic survival of NK cells to constitutively activated Ras in lymphoproliferative disease of granular lymphocytes (LDGL).

Chronic NK lymphoproliferative disease of large granular lymphocytes (LDGL) is characterized by the expansion of activated CD3-, CD16+ or CD56+ lymphocytes. The mechanism of survival of NK cells from LDGL patients is unknown but may be related to antigenic stimulation. There is currently no standard effective therapy for LDGL, and the disease is characteristically resistant to standard forms of chemotherapy. We found evidence of constitutive activation of extracellular-regulated kinase (ERK) in NK cells from 13/13 patients with NK-LDGL (one patient with aggressive and 12 patients with chronic disease). Ablation of ERK activity by inhibitors or a dominant-negative form of MEK, the upstream activator of ERK, reduced the survival of patient NK cells. Ras was also constitutively active in patient NK cells, and exposure of cells to the Ras inhibitor FTI2153 or to dominant-negative-Ras resulted not only in ERK inhibition but also in enhanced apoptosis in both the presence and absence of anti-Fas. Therefore, we conclude that a constitutively active Ras/MEK/ERK pathway contributes to the accumulation of NK cells in patients with NK-LDGL. These findings suggest that strategies to inhibit this signaling pathway may be useful for the treatment of the NK type of LDGL.

Dysregulated NK receptor expression in patients with lymphoproliferative disease of granular lymphocytes.

The natural killer (NK) type of lymphoproliferative disease of granular lymphocytes (LDGL) is associated with the expansion of CD3(-), CD16(+), and/or CD56(+) lymphocytes. We have examined the repertoire of NK receptors expressed on these cells and delineated the functional activity. We found skewed NK receptor expression on patient NK cells. Reactivity to a single anti-killer cell immunoglobulin-like receptor (anti-KIR) antibody was noted in 7 of 13 patients. LDGL patients variably expressed NKp30, NKp44, and NKp46 RNA. In contrast, CD94 and its inhibitory heterodimerization partner NKG2A were homogeneously expressed at high levels on these NK cells. Interestingly, these patients expressed a large number of activating KIR receptors by genotype analysis. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that lower than normal levels of RNA of the inhibitory KIR was present in some patients in contrast to normal NK cells. Consistent with a high level of activating receptors, we found the NK-LDGL cells have potent cytolytic function in both direct and redirected cytotoxicity assays. These results demonstrate that patients with NK-LDGL have an increased activating-to-inhibitory KIR ratio. This altered ratio might induce inappropriate lysis or cytokine production and impact the disease pathogenesis.