IFN-gamma priming of adipose-derived stromal cells at "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodeling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN-gamma on adipose tissue-derived MSCs (ASCs) at tissue-related O2 level (5%) and acute hypoxic stress (0.1% O2 ) were assessed as alterations of ASCs' CFU-F, proliferation, migration, osteo-commitment. IFN-gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2 -adapted ASCs. The prominent changes in ASC cytoskeleton-related gene transcription was detected. IFN-gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL-8, while MCP-1 and IL-6 were stimulated. Conditioned medium of IFN-gamma-primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in "wound closure." Short-term hypoxia suppressed CFU-F number, IFN-gamma-induced elevation of IL-6 and endothelial cell migration, while it abolished IFN-gamma-provoked VEGF inhibition. After N-acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL-6 and suppression of IL-8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN-gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodeling functions.

A Simple and User-Friendly Method for High-Quality Preparation of Pollen Grains for Scanning Electron Microscopy (SEM).

Pollen is becoming an increasingly important subject for molecular researchers in genetic engineering, plant breeding, and environmental monitoring. To broaden the scope of these studies, it is essential to develop accessible methods for scientists who are not specialized in palynology. The article presents a simplified technical procedure for preparing pollen grains for scanning electron microscopy (SEM). The protocol is convenient for any molecular laboratory due to its small set of reagents, ease of execution, low cost, does not require special equipment, and takes only one hour to complete. The high penetrating ability of formaldehyde and the final delicate dehydration using hexamethyldisilazane (HMDS) instead of critical point drying allow for sufficient preservation of the architecture of the aperture, which is considered a gateway for the passage of biomolecules. The method was successfully applied to pollen grains of representatives of dicotyledons (beetroot, petunia, radish, tomato and tobacco) and monocotyledons (lily, onion, corn, rye and wheat). Species studied included insect-pollinated (entomophilous) and wind-pollinated (anemophilous) species. A comparative analysis of the sizes of fresh living pollen grains under a light microscope and those prepared for SEM showed some shrinkage. Quantitative analysis of the degree of pollen grain shrinkage showed that this process depends on the initial shape of dry pollen grains, and the number and structure of apertures. The results support the theoretical model of the folding/unfolding pathways of pollen grains.

Pilot Study on Boiling Histotripsy Treatment of Human Leiomyoma Ex Vivo.

OBJECTIVE: As an alternative to surgical excision and magnetic resonance-guided thermal high-intensity focused ultrasound ablation of uterine leiomyoma, this work was aimed at pilot feasibility demonstration of use of ultrasound-guided boiling histotripsy for non-invasive non-thermal fractionation of human uterine leiomyoma ex vivo. METHODS: A custom-made sector ultrasound transducer of 1.5-MHz operating frequency and nominal f-number F# = 0.75 was used to produce a volumetric lesion (two layers of 5 × 5 foci with a 1 mm step) in surgically resected human leiomyoma ex vivo. A sequence of 10 ms pulses (P+/P-/As = 157/-25/170 MPa in situ) with 1% duty cycle was delivered N = 30 times per focus under B-mode guidance. The treatment outcome was evaluated via B-mode imaging and histologically with hematoxylin and eosin and Masson's trichrome staining. RESULTS: The treatment was successfully performed in less than 30 min and resulted in formation of a rectangular lesion visualized on B-mode images during the sonication as an echogenic region, which sustained for about 10 min post-treatment. Histology revealed loss of cellular structure, necrotic debris and globules of degenerated collagen in the target volume surrounded by injured smooth muscle cells. CONCLUSION: The pilot experiment described here indicates that boiling histotripsy is feasible for non-invasive mechanical disintegration of human uterine leiomyoma ex vivo under B-mode guidance, encouraging further investigation and optimization of this potential clinical application of boiling histotripsy.

Elastic Properties of Aging Human Hematoma Model In Vitro and Its Susceptibility to Histotripsy Liquefaction.

OBJECTIVE: Tissue susceptibility to histotripsy disintegration has been reported to depend on its elastic properties. This work was aimed at investigation of histotripsy efficiency for liquefaction of human hematomas, depending on their stiffness and degree of retraction over time (0-10 d). METHODS: As an in vitro hematoma model, anticoagulated human blood samples (200 mL) were recalcified at different temperatures. In one set of samples, the shear modulus was measured by shear wave elastography during blood clotting at 10℃, 22℃ and 37℃, and then daily during further aging. The ultrastructure of the samples was analyzed daily with scanning electron microscopy (SEM). Another set of blood samples (50-200 mL) were recalcified at 37℃ for density and retraction measurements over aging and exposed to histotripsy at varying time points. Boiling histotripsy (2.5 ms pulses) and hybrid histotripsy (0.2 ms pulses) exposures (2 MHz, 1% dc, P+/P-/As = 182/-27/207 MPa in situ) were used to produce either individual cigar-shaped or volumetric (0.8-3 mL) lesions in samples incubated for 3 h, 5 d and 10 d. The obtained lesions were sized, then the lysate aspirated under B-mode guidance was analyzed ultrastructurally and diluted in distilled water for sizing of residual fragments. RESULTS: It was found that clotting time decreased from 113 to 25 min with the increase in blood temperature from 10℃ to 37℃. The shear modulus increased to 0.53 ± 0.17 kPa during clotting and remained constant within 8 d of incubation at 2℃. Sample volumes decreased by 57% because of retraction within 10 d. SEM revealed significant echinocytosis but unchanged ultrastructure of the fibrin meshwork. Liquefaction rate and lesion dimensions produced with the same histotripsy protocols correlated with the increase in the degree of retraction and were lower in retracted samples versus freshly clotted samples. More than 80% of residual fibrin fragments after histotripsy treatment were shorter than 150 µm; the maximum length was 208 µm, allowing for unobstructed aspiration of the lysate with most clinically used needles. CONCLUSION: The results indicate that hematoma susceptibility to histotripsy liquefaction is not entirely determined by its stiffness, and correlates with the retraction degree.

Boiling Histotripsy in Ex Vivo Human Brain: Proof-of-concept.

OBJECTIVE: Non-invasive surgical approaches, including boiling histotripsy (BH), are currently being developed for the treatment of brain disorders aiming to avoid craniotomy and exposure of intervening tissues, and, thus, minimize associated complications. This work aimed to demonstrate the feasibility of BH for mechanical fractionation of human brain tissues ex vivo under B-mode guidance, with preliminary measurements of tissue stiffness via shear wave elastography. METHODS: Young's moduli of 25 human autopsy brain samples obtained from de-identified patients of 51-91 y old (median 77 y old) were measured via shear wave elastography prior to BH sonication. Seventeen volumetric BH lesions (1-4 layers of 5 × 5 points with a 1- mm step) were produced near brain surface (n = 10), in white matter (n = 3), in thalamus (n = 2), and globus pallidus (n = 2) using 12 element 1.5 MHz sector transducer under B-mode guidance with 10 ms or 2 ms pulses delivered 10 or 15 times per sonication point with 1% duty cycle. After treatment, the lesions were evaluated grossly through bisection, histologically with hematoxylin and eosin staining, and ultrastructurally via scanning and transmission electron microscopy. RESULTS: Young's moduli of autopsy brain samples were lower in older patients (from 32.9 ± 6.6 kPa in 51 y olds to 10 ± 2 kPa in 91 y olds) and at higher temperature (6%-50% lower at 37°Ð¡ vs 23°Ð¡), and were within the range observed clinically. All tested BH treatments performed near the brain surface (i.e., mostly in gray matter) resulted in formation of well-demarcated rectangular lesions with homogenized content and sharp boundaries, with majority of residual fragments below 100 microns. The use of shorter pulses (2 ms vs 10 ms) accelerated the treatment at least threefold, and the highest liquefaction rate was 568 mm3/min. White matter was more resistant to BH vs gray matter: at least 15 pulses of 2 ms duration were required per each sonication point, and the liquefaction rate was three times lower. The ability of BH to produce lesions in thalamus and globus pallidus was also confirmed. CONCLUSION: This work presents the first demonstration of BH proof-of-concept in human brain tissues ex vivo under B-mode guidance with clinically relevant treatment rates.

Hypoxic Extracellular Matrix Preserves Its Competence after Expansion of Human MSCs under Physiological Hypoxia In Vitro.

Tissue-relevant O2 levels are considered as an important tool for the preconditioning of multipotent mesenchymal stromal cells (MSCs) for regenerative medicine needs. The present study investigated the quality and functions of the extracellular matrix (ECM) of MSCs under low O2 levels. Human adipose tissue-derived MSCs were continuously expanded under normoxia (20% O2, N) or "physiological" hypoxia (5% O2, Hyp). Decellularized ECM (dcECM) was prepared. The structure of the dcECM was analyzed using confocal laser and scanning electron microscopy. Collagen, dcECM-N, and dcECM-Hyp were recellularized with MSC-N and further cultured at normoxia. The efficacy of adhesion, spreading, growth, osteogenic potential, and paracrine activity of recellularized MSC-N were evaluated. At low O2, the dcECM showed an increased alignment of fibrillar structures and provided accelerated spreading of MSC-N, indicating increased dcECM-Hyp stiffness. We described O2-dependent "ECM-education" of MSC-N when cultured on dcECM-Hyp. This was manifested as attenuated spontaneous osteo-commitment, increased susceptibility to osteo-induction, and a shift in the paracrine profile. It has been suggested that the ECM after physiological hypoxia is able to ensure the maintenance of a low-commitment state of MSCs. DcECM, which preserves the competence of the natural microenvironment of cells and is capable of "educating" others, appears to be a prospective tool for guiding cell modifications for cell therapy and tissue engineering.

Initial Assessment of Boiling Histotripsy for Mechanical Ablation of Ex Vivo Human Prostate Tissue.

Boiling histotripsy (BH) is a focused ultrasound technology that uses millisecond-long pulses with shock fronts to induce mechanical tissue ablation. The pulsing scheme and mechanisms of BH differ from those of cavitation cloud histotripsy, which was previously developed for benign prostatic hyperplasia. The goal of the work described here was to evaluate the feasibility of using BH to ablate fresh ex vivo human prostate tissue as a proof of principle for developing BH for prostate applications. Fresh human prostate samples (N = 24) were obtained via rapid autopsy (<24 h after death, institutional review board exempt). Samples were analyzed using shear wave elastography to ensure that mechanical properties of autopsy tissue were clinically representative. Samples were exposed to BH using 10- or 1-ms pulses with 1% duty cycle under real-time B-mode and Doppler imaging. Volumetric lesions were created by sonicating 1-4 rectangular planes spaced 1 mm apart, containing a grid of foci spaced 1-2 mm apart. Tissue then was evaluated grossly and histologically, and the lesion content was analyzed using transmission electron microscopy and scanning electron microscopy. Observed shear wave elastography characterization of ex vivo prostate tissue (37.9 ± 22.2 kPa) was within the typical range observed clinically. During BH, hyperechoic regions were visualized at the focus on B-mode, and BH-induced bubbles were also detected using power Doppler. As treatment progressed, hypoechoic regions of tissue appeared, suggesting successful tissue fractionation. BH treatment was twofold faster using shorter pulses (1 ms vs. 10 ms). Histological analysis revealed lesions containing completely homogenized cell debris, consistent with histotripsy-induced mechanical ablation. It was therefore determined that BH is feasible in fresh ex vivo human prostate tissue producing desired mechanical ablation. The study supports further work aimed at translating BH technology as a clinical option for prostate ablation.

Ultrastructural Analysis of Volumetric Histotripsy Bio-effects in Large Human Hematomas.

Large-volume soft tissue hematomas are a serious clinical problem, which, if untreated, can have severe consequences. Current treatments are associated with significant pain and discomfort. It has been reported that in an in vitro bovine hematoma model, pulsed high-intensity focused ultrasound (HIFU) ablation, termed histotripsy, can be used to rapidly and non-invasively liquefy the hematoma through localized bubble activity, enabling fine-needle aspiration. The goals of this study were to evaluate the efficiency and speed of volumetric histotripsy liquefaction using a large in vitro human hematoma model. Large human hematoma phantoms (85 cc) were formed by recalcifying blood anticoagulated with citrate phosphate dextrose/saline-adenine-glucose-mannitol solution. Typical boiling histotripsy pulses (10 or 2 ms) or hybrid histotripsy pulses using higher-amplitude and shorter pulses (0.4 ms) were delivered at 1% duty cycle while continuously translating the HIFU focus location. Histotripsy exposures were performed under ultrasound guidance with a 1.5-MHz transducer (8-cm aperture, F# = 0.75). The volume of liquefied lesions was determined by ultrasound imaging and gross inspection. Untreated hematoma samples and samples of the liquefied lesions aspirated using a fine needle were analyzed cytologically and ultrastructurally with scanning electron microscopy. All exposures resulted in uniform liquid-filled voids with sharp edges; liquefaction speed was higher for exposures with shorter pulses and higher shock amplitudes at the focus (up to 0.32, 0.68 and 2.62 mL/min for 10-, 2- and 0.4-ms pulses, respectively). Cytological and ultrastructural observations revealed completely homogenized blood cells and fibrin fragments in the lysate. Most of the fibrin fragments were less than 20 µm in length, but a number of fragments were up to 150 µm. The lysate with residual debris of that size would potentially be amenable to fine-needle aspiration without risk for needle clogging in clinical implementation.

Treating Porcine Abscesses with Histotripsy: A Pilot Study.

Infected abscesses are walled-off collections of pus and bacteria. They are a common sequela of complications in the setting of surgery, trauma, systemic infections and other disease states. Current treatment is typically limited to antibiotics with long-term catheter drainage, or surgical washout when inaccessible to percutaneous drainage or unresponsive to initial care efforts. Antibiotic resistance is also a growing concern. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. In particular, short pulses of focused ultrasound (i.e., histotripsy) generate mechanical damage through localized cavitation, representing a potential new paradigm for treating abscesses non-invasively, without the need for long-term catheterization and antibiotics. In this pilot study, boiling and cavitation histotripsy treatments were applied to subcutaneous and intramuscular abscesses developed in a novel porcine model. Ultrasound imaging was used to evaluate abscess maturity for treatment monitoring and assessment of post-treatment outcomes. Disinfection was quantified by counting bacteria colonies from samples aspirated before and after treatment. Histopathological evaluation of the abscesses was performed to identify changes resulting from histotripsy treatment and potential collateral damage. Cavitation histotripsy was more successful in reducing the bacterial load while having a smaller treatment volume compared with boiling histotripsy. The results of this pilot study suggest focused ultrasound may lead to a technology for in situ treatment of acoustically accessible abscesses.

Pilot in vivo studies on transcutaneous boiling histotripsy in porcine liver and kidney.

Boiling histotripsy (BH) is a High Intensity Focused Ultrasound (HIFU) method for precise mechanical disintegration of target tissue using millisecond-long pulses containing shocks. BH treatments with real-time ultrasound (US) guidance allowed by BH-generated bubbles were previously demonstrated ex vivo and in vivo in exposed porcine liver and small animals. Here, the feasibility of US-guided transabdominal and partially transcostal BH ablation of kidney and liver in an acute in vivo swine model was evaluated for 6 animals. BH parameters were: 1.5 MHz frequency, 5-30 pulses of 1-10 ms duration per focus, 1% duty cycle, peak acoustic powers 0.9-3.8 kW, sonication foci spaced 1-1.5 mm apart in a rectangular grid with 5-15 mm linear dimensions. In kidneys, well-demarcated volumetric BH lesions were generated without respiratory gating and renal medulla and collecting system were more resistant to BH than cortex. The treatment was accelerated 10-fold by using shorter BH pulses of larger peak power without affecting the quality of tissue fractionation. In liver, respiratory motion and aberrations from subcutaneous fat affected the treatment but increasing the peak power provided successful lesion generation. These data indicate BH is a promising technology for transabdominal and transcostal mechanical ablation of tumors in kidney and liver.

Mechanical decellularization of tissue volumes using boiling histotripsy.

High intensity focused ultrasound (HIFU) is rapidly advancing as an alternative therapy for non-invasively treating specific cancers and other pathological tissues through thermal ablation. A new type of HIFU therapy-boiling histotripsy (BH)-aims at mechanical fractionation of into subcellular fragments, with a range of accompanying thermal effects that can be tuned from none to substantial depending on the requirements of the application. The degree of mechanical tissue damage induced by BH has been shown to depend on the tissue type, with collagenous structures being most resistant, and cellular structures being most sensitive. This has been reported for single BH lesions, but has not been replicated in large volumes. Such tissue selectivity effect has potential uses involving tissue decellularization for biofabrication technologies as well as mechanical ablation by BH while sparing critical structures. The goal of this study was to investigate tissue decellularization effect in larger, clinically relevant liquefied volumes of tissue, and to evaluate the accumulated thermal effect in the volumetric lesions under different exposure parameters. All BH exposures were performed with a 256-element 1.2 MHz array of a magnetic resonance imaging-guided HIFU (MR-HIFU) clinical system (Sonalleve V1, Profound Medical Inc, Mississauga, Canada). The volumetric BH lesions were produced in degassed ex vivo bovine liver using 1-10 ms long pulses with in situ shock amplitudes of 75-100 MPa at the focus and pulse repetition frequencies (PRFs) of 1-10 Hz covering a range of effects from pure mechanical homogenization to thermal ablation. Multimodal analysis of the lesions was then performed, including microstructure (histological), ultrastructure (electron microscopy), and molecular (biochemistry) methods. Results show a range of tissue effects in terms of the degree of tissue selectivity and the amount of heat generated in large BH lesions, thereby demonstrating potential for treatments tailored to different clinical applications.

Inactivation of Planktonic Escherichia coli by Focused 1-MHz Ultrasound Pulses with Shocks: Efficacy and Kinetics Upon Volume Scale-Up.

This study addresses inactivation of E. coli in either 5- or 10-mL volumes, which were 50- to 100-fold greater than used in an earlier study (Brayman et al. 2017). Cells were treated with 1-MHz pulsed high-intensity focused ultrasound (10 cycles, 2-kHz repetition frequency, +65/-12.8 MPa focal pressures). The surviving fraction was assessed by coliform assay, and inactivation demonstrated curvilinear kinetics. The reduction of surviving fraction to 50% required 2.5 or 6 min in 5- or 10-mL samples, respectively. Exposure of 5 mL for 20 min reduced the surviving fraction to ∼1%; a similar exposure of 10-mL samples reduced the surviving fraction to ∼10%. Surviving cells from 5-min exposures appeared normal under light microscopy, with minimal debris; after 20 min, debris dominated. Transmission electron microscopy images of insonated samples showed some undamaged cells, a few damaged but largely intact cells and comminuted debris. Cellular damage associated with substantive but incomplete levels of inactivation can be variable, ranging from membrane holes tens of nanometers in diameter to nearly complete comminution.

Molecular Bases of Brain Preconditioning.

Preconditioning of the brain induces tolerance to the damaging effects of ischemia and prevents cell death in ischemic penumbra. The development of this phenomenon is mediated by mitochondrial adenosine triphosphate-sensitive potassium ([Formula: see text]) channels and nitric oxide signaling (NO). The aim of this study was to investigate the dynamics of molecular changes in mitochondria after ischemic preconditioning (IP) and the effect of pharmacological preconditioning (PhP) with the [Formula: see text]-channels opener diazoxide on NO levels after ischemic stroke in rats. Immunofluorescence-histochemistry and laser-confocal microscopy were applied to evaluate the cortical expression of electron transport chain enzymes, mitochondrial [Formula: see text]-channels, neuronal and inducible NO-synthases, as well as the dynamics of nitrosylation and nitration of proteins in rats during the early and delayed phases of IP. NO cerebral content was studied with electron paramagnetic resonance (EPR) spectroscopy using spin trapping. We found that 24 h after IP in rats, there is a two-fold decrease in expression of mitochondrial [Formula: see text]-channels (p = 0.012) in nervous tissue, a comparable increase in expression of cytochrome c oxidase (p = 0.008), and a decrease in intensity of protein S-nitrosylation and nitration (p = 0.0004 and p = 0.001, respectively). PhP led to a 56% reduction of free NO concentration 72 h after ischemic stroke simulation (p = 0.002). We attribute this result to the restructuring of tissue energy metabolism, namely the provision of increased catalytic sites to mitochondria and the increased elimination of NO, which prevents a decrease in cell sensitivity to oxygen during subsequent periods of severe ischemia.

Microbiota-related Changes in Bile Acid & Tryptophan Metabolism are Associated with Gastrointestinal Dysfunction in a Mouse Model of Autism.

Autism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental conditions worldwide. There is growing awareness that ASD is highly comorbid with gastrointestinal distress and altered intestinal microbiome, and that host-microbiome interactions may contribute to the disease symptoms. However, the paucity of knowledge on gut-brain axis signaling in autism constitutes an obstacle to the development of precision microbiota-based therapeutics in ASD. To this end, we explored the interactions between intestinal microbiota, gut physiology and social behavior in a BTBR T+Itpr3tf/J mouse model of ASD. Here we show that a reduction in the relative abundance of very particular bacterial taxa in the BTBR gut - namely, bile-metabolizing Bifidobacterium and Blautia species, - is associated with deficient bile acid and tryptophan metabolism in the intestine, marked gastrointestinal dysfunction, as well as impaired social interactions in BTBR mice. Together these data support the concept of targeted manipulation of the gut microbiota for reversing gastrointestinal and behavioral symptomatology in ASD, and offer specific plausible targets in this endeavor.

Acute Hypoxic Stress Affects Migration Machinery of Tissue O2-Adapted Adipose Stromal Cells.

The ability of mesenchymal stromal (stem) cells (MSCs) to be mobilised from their local depot towards sites of injury and to participate in tissue repair makes these cells promising candidates for cell therapy. Physiological O2 tension in an MSC niche in vivo is about 4-7%. However, most in vitro studies of MSC functional activity are performed at 20% O2. Therefore, this study focused on the effects of short-term hypoxic stress (0.1% O2, 24 h) on adipose tissue-derived MSC motility at tissue-related O2 level. No significant changes in integrin expression were detected after short-term hypoxic stress. However, O2 deprivation provoked vimentin disassembly and actin polymerisation and increased cell stiffness. In addition, hypoxic stress induced the downregulation of ACTR3, DSTN, MACF1, MID1, MYPT1, NCK1, ROCK1, TIAM1, and WASF1 expression, the products of which are known to be involved in leading edge formation and cell translocation. These changes were accompanied by the attenuation of targeted and nontargeted migration of MSCs after short-term hypoxic exposure, as demonstrated in scratch and transwell migration assays. These results indicate that acute hypoxic stress can modulate MSC function in their native milieu, preventing their mobilisation from sites of injury.

Mechanical characteristics of mesenchymal stem cells under impact of silica-based nanoparticles.

Silica-based nanoparticles (NPs) pose great potential for medical and biological applications; however, their interactions with living cells have not been investigated in full. The objective of this study was to analyze the mechanical characteristics of mesenchymal stem cells when cultured in the presence of silica (Si) and silica-boron (SiB) nanoparticles. Cell stiffness was measured using atomic force microscopy; F-actin structure was evaluated using TRITC-phalloidin by confocal microscopy. The obtained data suggested that the cell stiffness increased within the following line: 'Control' - 'Si' - 'SiB' (either after 1-h cultivation or 24-h incubation). Moreover, the cell stiffness was found to be higher after 1-h cultivation as compared to 24-h cultivation. This result shows that there is a two-phase process of particle diffusion into cells and that the particles interact directly with the membrane and, further, with the submembranous cytoskeleton. Conversely, the intensity of phalloidin fluorescence dropped within the same line: Control - Si - SiB. It could be suggested that the effects of silica-based particles may result in structural reorganization of cortical cytoskeleton with subsequent stiffness increase and concomitant F-actin content decrease (for example, in recruitment of additional actin-binding proteins within membrane and regrouping of actin filaments).

Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors.

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O2. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34+ cells up to 6 (20% O2) and 8 (5% O2) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O2, than in standard cultivation (20% O2). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O2. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34+ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O2 levels at the expense of direct and paracrine cell-to-cell interactions.