Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors

The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages

Differentiation of murine embryonic cells towards the haematopoietic cell lineage using the HepG2 conditioned medium and encapsulation in a rotating wall vessel bioreactor

Embryonic stem cells (ESCs) are known for their unique property to be maintained almost indefinitely in an undifferentiated, proliferating state with the potential to give rise to all types of cells. Current established protocols for the culture and differentiation of ESCs are cumbersome and inefficient involving three stages: a) maintenance or expansion of undifferentiated ESCs, b) spontaneous differentiation through formation of embryoid bodies (EBs), and c) dissociation of the EBs and replating leading to the terminal differentiation to the desired lineages. One of the major challenges in the use of ESCs for cell therapy is controlling their differentiation pathways. Optimal culture conditions and requirement as well as precise differentiation mechanisms and cellular interactions within EBs are still not well characterised resulting in sub-optimal control of homogenous differentiation especially due to the formation of all three germ layers. Attempts on developing an efficient culture protocol have been widely reported in order to overcome the limitations. Recent research approaches have shown that treatment with conditioned medium derived from HepG2, a human hepatocarcinoma cell line enhances the formation of multipotent mesodermal progenitors from ESCs. This promotes a greater control of ESC differentiation in a lineage-specific fashion possibly resulting in efficient haematopoietic differentiation. In this study, we have developed an integrated, single step bioprocess for ESCs hematopoietic differentiation that: a) uses HepG2-conditioned medium (HepG2-CM), that stimulates mesoderm formation, b) facilitates three dimensional (3D) culture through encapsulation of undifferentiated ESCs in hydrogels, c) bypasses EB formation, and d) involves culture in a rotating wall vessel bioreactor that does not require passaging of the cells and is scalable and automatable. In conclusion, this thesis reports the development of a novel culture system for the efficient single-step haematopoietic differentiation of ESC resulting in a reproducible, scalable, high-intensity culture system of mESCs for ex-vivo blood manufacture

Cell extract-based reprogramming of somatic cells

The differentiation potential of adult stem cells (ASC) has long been thought to be limited to cell lineages present in the organ from which they are derived; however, several studies have challenged this notion by demonstrating that some ASC exhibit a remarkably high degree of plasticity. Unlike terminally differentiated somatic cells, the less differentiated state of ASC can assume the functional phenotypes and expression profiles of cells unique to other tissues. The expansive repertoire of differentiation potential exhibited by ASC suggests these cells possess characteristics similar to pluripotent cells, including epigenomic regulatory pattern. Therefore, ASC may be better equipped for complete epigenetic reprogramming than terminally differentiated cells. The objective of Experiment 1 was to analyze bovine adipose-derived adult stem cells (ADAS) and fetal fibroblast (BFF) cells for the presence of the pluripotency-associated genes, Oct-4, Nanog, and Sox-2. Because the endogenous expression of these genes is believed to contribute to reprogramming efficiency, Experiment 2 sought to increase Oct-4, Nanog and Sox-2 expression levels in BFF cells through exposure to ADAS cell extracts. Transcripts for all three pluripotency-associated genes were detected in all BFF and ADAS cell samples at every passage analyzed; however, expression was quite low and highly variable between cell lines and passage numbers. Nevertheless, these findings support the notion that these cells are less differentiated than other somatic cells. This less differentiated state appears to sufficient for at least the partial reprogramming of BFF cells using ADAS cell extracts in a cell extract-based nuclear reprogramming system

Antisenescence Effect of REAC Biomodulation to Counteract the Evolution of Myelodysplastic Syndrome

About 30 percent of patients diagnosed with myelodysplastic syndromes (MDS) progress to acute myeloid leukemia (AML). The senescence of bone marrow‐derived mesenchymal stem cells (BMSCs) seems to be one of the determining factors in inducing this drift. Research is continuously looking for new methodologies and technologies that can use bioelectric signals to act on senescence and cell differentiation towards the phenotype of interest. The Radio Electric Asymmetric Conveyer (REAC) technology, aimed at reorganizing the endogenous bioelectric activity, has already shown to be able to determine direct cell reprogramming effects and counteract the senescence mechanisms in stem cells. Aim of the present study was to prove if the anti-senescence results previously obtained in different kind of stem cells with the REAC Tissue optimization – regenerative (TO-RGN) treatment, could also be observed in BMSCs, evaluating cell viability, telomerase activity, p19ARF, P21, P53, and hTERT gene expression. The results show that the REAC TORGN treatment may be a useful tool to counteract the BMSCs senescence which can be the basis of AML drift. Nevertheless, further clinical studies on humans are needed to confirm this hypothesis

Human Hair Keratin Protein, Hair Fibers and Hydroxyapatite (HA) Composite Scaffold for Bone Tissue Regeneration

The field of tissue engineering aims at promoting the regeneration of tissues or replacement of failing or malfunctioning tissue by means of combining a scaffold material, adequate cells and bioactive molecules. Different materials have been proposed for use as three-dimensional porous scaffolds for bone tissue engineering procedures. Among them, polymers of natural origin are one of the most attractive options mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this study, two biocompatible composite scaffolds were developed from natural polymer by tissue engineering approach and tested in vitro. The first Scaffold (SCAF-1) that was developed was composed of human hair keratin protein and human hair fibers (cuticle-cortex). The second scaffold (SCAF-2) was composed of human hair keratin protein, human hair fibers (cuticle-cortex) and hydroxyapatite (HA) particles. SEM and EDX were used to analyze the three dimensional structure, surface chemistry and pore size of the scaffolds. Both scaffolds showed a three-dimensional structure with a pore size ranging from 40-500℗æm and porosity greater than 50 . Compressive tests were carried out under dry as well as wet conditions for both scaffolds. SCAF-1 showed compressive modulus of 0.009 MPa in wet condition and 0.90 MPa in a dry condition. Likewise, SCAF-2 had compressive modulus of 0.09 MPa in wet condition and 2.7 MPa in dry condition. Cell culture experiments with bone marrow stromal cells demonstrate that the composite scaffolds support cell attachment and proliferation.Overall, human hair keratin scaffolds have been shown to have a porous three-dimensional structure that induces proliferation of GFP- stromal cells for bone tissue regeneration. These preliminary results suggest that human hair keratin, cuticle-cortex fibers and HA composite scaffolds appear to be an interesting structure for potential studies in bone tissue engineerin

Eliminating Acute Myeloid Leukemia Stem Cells by Targeting the Niche Microenviromnent: Co-Inhibition of TNF/IL1- JNK and NF-κb

Leukemia Stem Cells (LSCs) from Acute Myeloid Leukemia (AML) require the activity of the transcription factor NF-kB to maintain stemness and drive tumor formation. Blocking NF-kB can preferentially eliminate LSCs in vitro with minimal effects on healthy Hematopoietic Stem and Progenitor Cells (HSPCs), making NF-kB a compelling target for anti-leukemia therapies. However, blocking NF-kB in vivo can only extend survival for a short period of time before transplanted mice succumb to the disease. I propose this is due to components of the in vivo niche supporting LSC survival and compensating for the inhibition of NF-kB. I observed patients with partially differentiated blast-like AML (subtypes M3, M4, and M5) have significantly elevated levels of pro-inflammatory cytokines TNFa and IL-1a/b; (TNF and IL1) circulating in their peripheral blood. Further study revealed these cytokines are primarily produced by LSCs because such cells express several times more of TNF and IL1 than their healthy tissue counterparts. I found that TNF and IL1 stimulate the growth and expansion of LSCs while inducing cell death in HSPCs. Also, LSC-conditioned media alone is sufficient to drive apoptosis/necroptosis in HSPCs that can be prevented by blocking TNF and IL1, suggesting a mechanism for hematopoietic repression commonly observed in AML cases. TNF and IL1 drive their pro-inflammatory effects on target cells through activation of cellular signaling networks. Both TNF and IL1 are potent activators of NF-kB in almost all cells studied, connecting the ability of these cytokines to drive LSC growth with the need of NF-kB for survival. In addition, TNF and IL1 also stimulate activation of JNK signaling which operates in parallel to NF-kB in LSCs and HSPCs. I found that JNK stimulation results in cell death in HSPCs by subsequent inactivation of pro-survival Bcl-2 family proteins by phosphorylation. However, LSCs convert JNK-mediated cell death signals into proliferation/survival signals by both limiting the signal duration to \u3c60 min through dephosphorylation by Map Kinase Phosphatase 5, and also by upregulating JNK\u27s nuclear target c-Jun. Such short duration of JNK activation correlates to activation of JNK\u27s nuclear targets without activating the death signal. I determined that concurrent inhibition of NF-kB and JNK has two major effects: (1) combined inhibition specifically targets LSCs in vitro and in vivo, and (2) the toxicity in healthy HSPCs due to loss of NF-kB signaling is mediated by JNK, making the combined treatment protective. I can substantially increase survival in AML-transplanted mice if they are treated with combination of NF-kB and JNK inhibition in vivo. I can further extend survival in leukemic mice when I treat with blockers to upstream pro-inflammatory cytokines: anti-TNF, anti-IL1, and NF-kB inhibitor. These data provide a strong rationale to treat AML patients by combined inhibition of both TNF/IL1- JNK and NF-kB signaling

The Role of Osteomacs in Regulating Stem Cell Function and the Hematopoietic Niche

Indiana University-Purdue University Indianapolis (IUPUI)Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). Reported here is a detailed characterization of OM and description of discriminating phenotypic and functional properties that clearly distinguish OM from bone marrow-derived macrophages (BM Mφ). Furthermore, it was established that OM support hematopoiesis enhancing activity of osteoblasts and that this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing OM, osteoblasts and megakaryocytes. Interestingly, BM Mφ were unable to mediate the same hematopoiesis enhancing activity regardless of whether megakaryocytes were present in co-culture or not. Subsequently, to understand the importance of networking between the residents of the niche, 3D tissue cytometry was performed on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, osteoblasts, megakaryocytes and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. In addition, single cell mRNAseq and CyTOF was performed to assess genetic and proteomic expression changes in OM following their interaction with megakaryocytes. These studies revealed the upregulation of CD166 and embigin on OM via osteoblast and megakaryocyte interactions. Clonogenic assays were conducted to examine the impact of these molecules in hematopoietic function. When these assays were initiated with CD166 KO OM or shRNA-mediated embigin knockdown OM, it was established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. This data, for the first time, reveal intimate spatial interactions between OM, osteoblasts, megakaryocytes and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, osteoblasts and megakaryocytes and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function.2020-09-1

Chondro progenitor cell response to specifically modified substrate interfaces

Current visions in cartilage repair aim at moving from fibrocartilaginous to a more hyaline like repair tissue by combining autologous cells of different origins (in situ recruited or in vitro precultivated) with repair supporting biomaterials. The role of a chondro-supportive biomaterial within a cartilage defect is seen to support infiltration/recruitment of chnodroprogenitor cells (CPC), accelerate their chondrogenic differentiation and to protect/modulate the newly formed tissue. Overall, this thesis aimed at studying whether modification of selected substrate interface properties allows for modulating chondroprogenitor cell phenotype & function under expansion or differentiation conditions in vitro. The goal was to contribute to the definition of material characteristics which could be implemented in the design of biomaterials in order to improve current matrix assisted cartilage repair strategies and outcomes. Substrate composition & architecture (Chapter I) were found to modulate the chondrogenic differentiation of mesenchymal stem cells (MSC). Using a di block copolymer model substrate (Polyactive®) with a more hydrophobic composition better supported MSC chondrogenesis, than a more hydrophilic composition. Moreover, a highly interconnected 3D fibre deposited scaffold architecture allowed for the formation of larger MSC aggregates and was found to considerably better support MSC chondrogenesis than compression molded scaffolds. Restricting cell/substrate interaction specifically to an RGD-containing peptide ligand (Chapter II) modulated the de-differentiation of proliferating HAC and their subsequent capacity to form cartilaginous matrix. This demonstrated the advantage of small ECM fragments in combination with protein resistant materials to control cell/surface interaction. An important finding was the better maintenance of the HAC chondrocytic phenotype during expansion. It suggests, that an RGD-restricted substrate has the potential to improve the outcome of matrix assisted in situ cartilage repair, which initially requires recruited/infiltrated CPC to proliferate, while keeping/inducing their capacity to form cartilaginous matrix. Substrate elasticity allowed for modulating the chondrogenic commitment of HAC (Chapter III). The finding, that a soft substrate (0.3kPa) better supported the chondrogenic phenotype of HAC than i.e. a stiffer substrate (75kPa) suggests this parameter to be promising for modulating the outcome of matrix assisted cartilage repair. Overall, this thesis demonstrates that substrate properties hold substantial potential to modulate CPC behaviour, which could be exploited to improve materials employed in matrix assisted cartilage repair. Yet, although differentially supporting CPC chondrogenesis, none of the substrates was per se chondro-inductive (see chapter I and III) but required for additional, exogenic stimuli as for e.g. transforming growth factor beta (TGF). Thus, modulatory substrate properties as i.e. architecture, composition, ligand presentation and stiffness should be combined with the instructive capacity of soluble stimuli to exploit the full potential of biomaterials in cartilage repair