Endothelial Progenitor Research Articles

Cardioimmunology in Health and Diseases: Impairment of the Cardio-Spleno-Bone Marrow Axis Following Myocardial Infarction in Diabetes Mellitus.

A comprehensive understanding of the cardio-spleen-bone marrow immune cell axis is essential for elucidating the alterations occurring during the pathogenesis of diabetes mellitus (DM). This study investigates the dynamics of immune cell kinetics in DM after myocardial infarction (MI) over time. MI was induced in diabetic and healthy control groups using C57BL/N6 mice, with sacrifices occurring at days 1, 3, 7, and 28 post-MI to collect heart, peripheral blood (PB), spleen, and bone marrow (BM) samples. Cell suspensions from each organ were isolated and analyzed via flow cytometry. Additionally, the endothelial progenitor cell-colony-forming assay (EPC-CFA) was performed using mononuclear cells derived from BM, PB, and the spleen. The results indicated that, despite normal production in BM and the spleen, CD45+ cells were lower in the PB of DM mice at days 1 to 3. Further analysis revealed a reduction in total and pro-inflammatory neutrophils (N1s) in PB at days 1 to 3 and in the spleen at days 3 to 7 in DM mice, suggesting that DM-induced alterations in splenic neutrophils fail to meet the demand in PB and ischemic tissues. Infiltrating macrophages (total, M1, M2) were reduced at day 3 in the DM-ischemic heart, with total and M1 (days 1-3) and M2 (days 3-7) macrophages being significantly decreased in DM-PB compared to controls, indicating impaired macrophage recruitment and polarization in DM. Myeloid dendritic cells (mDCs) in the heart were higher from days 1 to 7, which corresponded with the enhanced recruitment of CD8+ cells from days 1 to 28 in the DM-infarcted myocardium. Total CD4+ cells decreased in DM-PB at days 1 to 3, suggesting a delayed adaptive immune response to MI. B cells were reduced in PB at days 1 to 3, in myocardium at day 3, and in the spleen at day 7, indicating compromised mobilization from BM. EPC-CFA results showed a marked decrease in definitive EPC colonies in the spleen and BM from days 1 to 28 in DM mice compared to controls in vitro, highlighting that DM severely impairs EPC colony-forming activity by limiting the differentiation of EPCs from primitive to definitive forms. Taking together, this study underscores significant disruptions in the cardio-spleen-bone marrow immune cell axis following MI in DM, revealing delayed innate and adaptive immune responses along with impaired EPC differentiation.

Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

BackgroundTransplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34+ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34+ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34+ isolated from cord blood (CB-CD34+) or fetal liver (FL-CD34+ and FL-CD34+CD14−) and their engraftment into immunocompromised NOD/Shi-scid Il2rgnull (NOG) mice.MethodsNOG mice were transplanted with either CB-CD34+, FL-CD34+ or FL-CD34+CD14− to generate CB-NOG, FL-NOG and FL-CD14−-NOG, respectively. After 15–20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood and several organs. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs. FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles.ResultsWe show that CB-CD34+ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34+ due to faster B cell development. However, upon long-term engraftment, FL-NOG display increased numbers of neutrophils, dendritic cells and macrophages in multiple tissues. In addition to HSPC, FL-CD34+ isolates contain non-hematopoietic CD14+ endothelial cells that enhance the engraftment of the human immune system in FL-NOG mice. We demonstrate that these CD14+CD34+ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14+CD34+ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection.ConclusionOur results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.

Abstract 1064: Creation Of Lymphatic Endothelial Cells From Induced Pluripotent Stem Cells For Use In Regenerative Therapy For Lymphedema

Background/Objective: Lymphedema is a condition characterized by impaired flow of interstitial fluid through the lymphatic system and chronic obliteration of distal lymphatic collecting vessels. Despite advances in our knowledge of the development of lymphatic vessels, there has been little progress translating this knowledge into clinically relevant regenerative therapies. Although lymphatic endothelial cells (LECs) share a lineage with arterial and venous endothelial cell progenitors, currently described processes to derive LECs from stem cells in vitro take up to a month, require embryoid body formation, and have been minimally characterized. Methods: We have developed a protocol for the generation of lymphatic endothelial cells from induced pluripotent stem cells (iPSCs) over 11 days. First, iPSCs are directed to become endothelial cell progenitors over 6 days and purified with CD144 magnetic beads. Then, ECs are directed to differentiate into LECs using a 5-day treatment with 10 ng/mL Ang1 alone or in combination with 200 ng/mL VEGFc. Human primary LECs were used as a positive control and iPSC-ECs without Ang1 or VEGFc treatment as well as fibroblasts were negative controls. Results: Treating iPSC-derived endothelial cells for 5 days with Ang1 +/- VEGFc yields iPSC-LECs with morphological similarity to primary lymphatic controls and expression of multiple lymphatic markers including Podoplanin, Lyve1, Prox1, and VEGFR3, verified by immunofluorescent staining, western blotting, and flow cytometry across two independent cell lines. In flow cytometry studies, 75% of LECs treated with Ang1 and 72% of LECs treated with Ang1 and VEGFc co-express Podoplanin and Lyve1. There was more than a three-fold increase compared to CD144+ endothelial progenitors (20.4%, p<0.001) while fibroblast expression was negligible (0.6%, p<0.001). Finally, the overall transcriptome of LECs treated with Ang1 was compared to primary human lymphatic cells using single cell RNA sequencing. Conclusions: Treatment with Ang1 with or without VEGFc allows fast and efficient generation of LECs from iPSCs. This allows LEC differentiation in vitro from a wide variety of patient specific cell types and improves the practicality of regenerative therapy for lymphedema.

Abstract 2147: Impact Of Donor Age On Microvasculature Self-assembly Of Induced Pluripotent Stem Cell Derived Endothelial Progenitors

Introduction: Current vascular tissue-engineering using induced pluripotent stem cells (iPSCs) predominantly relies on somatic cells obtained from newborn or fetal donors. However, iPSCs sourced from aged individuals exhibit epigenetic differences in angiogenic genes that may hinder their utility in iPSC derived vasculature. We hypothesized that, due to these differences, iPSCs from aged donors will exhibit decreased vasculogenic potential. Methods: We differentiated iPSCs into endothelial progenitors (iEPs) and encapsulated them in an interpenetrating polymer network hydrogel containing norbornene-functionalized hyaluronic acid and Collagen I at cell densities and hydrogel stiffness optimal for vasculogenesis. Seven days after encapsulation, the hydrogels were fixed, and the resulting microvasculature imaged. We then utilized a previously developed computational pipeline to quantify several vasculogenic properties. Results: We derived iEPs from four separate iPSC lines: 2 sourced from neonatal donors (ND) and 2 from mature donors (MD). Irrespective of donor age, differentiation yielded ~15% (n=4) iEPs of the total cell population. The two ND lines developed a similar plexus, whereas this structure was diminished in one adult line and absent in the second. Between iEPs matched for sex and somatic cell origin, there is a 50% decrease in vessel volume fraction and number of links between vessels, a 66% reduction in the percentage of vessels connected to the largest vessel, and a 43% reduction in branch points between vessels ( Fig. 1 ). Conclusion: Overall, these data suggest MD iEPs are less likely to form dense interconnected microvasculature as compared to ND iEPs. Interestingly, the average diameter did not change across all cell lines. This suggests that while MD iEPs exhibited cell clustering, they were less likely to branch out and interconnect with other clusters of iPSC-EPs to form interconnected networks

Abstract 136: Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk RMS has not increased above 20% in the last three decades. A deeper understanding of the basic biology driving RMS tumorigenesis is needed to discover novel therapeutic targets. RMS can be divided into two major histologic subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS accounts for 20% of pediatric cases and has poorer survival. The majority of ARMS tumors harbor t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. ARMS tumors that lack oncofusions more closely resemble ERMS. Classifying on fusion status more accurately describes the disease and highlights the significance of P3/7F. ARMS tumor genomes only contain an average of 6.4 somatic mutations suggesting the oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. RMS is commonly thought to arise from skeletal muscle progenitor cells that fail to terminally differentiate. However, RMS occurs throughout the body in sites devoid of skeletal muscle, underscoring the potential for alternative cells of origin. Our lab established that endothelial progenitors are indeed a cell of origin for ARMS by generating a genetically engineered mouse model with endothelial cell specific P3F expression coupled with Cdkn2a loss. We further generated a human model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which we direct differentiation to endothelial cell fate and then force P3F expression. P3F expression during endothelial cell directed differentiation blocked endothelial maturation and instead reprogrammed cells shifting cell fate to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the kinetics and specific genomic contributions facilitated by P3F during RMS transformation. To investigate the genomic changes occurring during iARMS transformation I utilized CUT&RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These results define the fundamental genomic changes needed for transformation into FP-RMS by P3F and uncovers the key determinants of ARMS cell identity. The iARMS system provides a unique platform to uncover how P3F establishes and maintains RMS cell state and to dissect the specific dependencies required to maintain the ARMS cell state. Understanding the fundamental mechanism of P3F-driven tumorigenesis could provide greater resolution into the key disease determinants to focus pre-clinical efforts on pivotal targets. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Brian J. Abraham, Mark E. Hatley. Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 136.

Direct programming of human pluripotent stem cells into endothelial progenitors with SOX17 and FGF2

Transcription factors (TFs) are pivotal in guiding stem cell behavior, including their maintenance and differentiation. Using single-cell RNA sequencing, we investigated TFs expressed in endothelial progenitors (EPs) derived from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Conversely, SOX17 knockdown via CRISPR-Cas13d significantly compromised EP differentiation. Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31- cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into endothelial cells. These findings underscore an undiscovered role of SOX17 in programming hPSCs toward an EP lineage, illuminating pivotal mechanisms in EP differentiation.

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor β (TGFβ) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

Small volume bone marrow aspirates with high progenitor cell concentrations maximize cell therapy dose manufacture and substantially reduce donor hemoglobin loss

BackgroundBone marrow (BM) transplantation is a life-saving therapy for hematological diseases, and the BM harbors also highly useful (progenitor) cell types for novel cell therapies manufacture. Yet, the BM collection technique is not standardized.MethodsBenchmarking our collection efficiency to BM collections worldwide (N = 1248), we noted a great variability of total nucleated cell (TNC) yields in BM products (HPC-M) with superior performance of our center, where we have implemented a small volume aspirate policy. Thus, we next prospectively aimed to assess the impact of BM collection technique on HPC-M quality. For each BM collection (N = 20 donors), small volume (3 mL) and large volume (10 mL) BM aspirates were sampled at 3 time points and analyzed for cell composition.ResultsCompared to large volume aspirates, small volume aspirates concentrated more TNCs, immune cells, platelets, hematopoietic stem/progenitor cells, mesenchymal stromal cells (MSCs), and endothelial progenitors. Inversely, the hemoglobin concentration was higher in large volume aspirates indicating more hemoglobin loss. Manufacturing and dosing scenarios showed that small volume aspirates save up to 42% BM volume and 44% hemoglobin for HPC-M donors. Moreover, MSC production efficiency can be increased by more than 150%.ConclusionsWe propose to consider small volume BM aspiration as standard technique for BM collection.

Posterior corneoscleral limbus: Architecture, stem cells, and clinical implications.

The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Fluidic Device System for Mechanical Processing and Filtering of Human Lipoaspirate Enhances Recovery of Mesenchymal Stem Cells.

Adipose tissue is an easily accessible source of stem and progenitor cells that offers exciting promise as an injectable autologous therapeutic for regenerative applications. Mechanical processing is preferred over enzymatic digestion, and the most common method involves shuffling lipoaspirate between syringes and filtering to produce nanofat. Although nanofat has shown exciting clinical results, the authors hypothesized that new device designs could enhance recovery of stem/progenitor cells through optimization of fluid dynamics principles, integration, and automation. The authors designed and fabricated the emulsification and micronization device (EMD) and the filtration device (FD) to replace the manual nanofat procedures. Using human lipoaspirate samples, the EMD and the FD were optimized and compared to traditional nanofat using ex vivo measurements of cell number, viability, and percentage of mesenchymal stem cells and endothelial progenitor cells. The EMD produced results statistically similar to nanofat, and these findings were confirmed for a cohort of diabetic patients. Combining the FD with the EMD was superior to manually filtered nanofat in terms of both recovered cell percentages (>1.5-fold) and numbers (two- to three-fold). Differences were statistically significant for total mesenchymal stem cells and a DPP4 + /CD55 + subpopulation linked to improved wound healing in diabetes. The new EMD and the FD improved mechanical processing of human lipoaspirate in terms of mesenchymal stem cell enrichment and number compared to traditional nanofat. Future work will seek to investigate the wound healing response both in vitro and in vivo, and to refine the technology for automated operation within clinical settings. The new devices improved mechanical processing of human lipoaspirate in terms of stem cell enrichment and number compared to traditional methods. Future work will seek to validate wound healing response and refine the technology for automated operation within clinical settings.

Targeting Hydrogen Sulfide Modulates Dexamethasone-Induced Muscle Atrophy and Microvascular Rarefaction, through Inhibition of NOX4 and Induction of MGF, M2 Macrophages and Endothelial Progenitors.

Long-term use of Glucocorticoids produces skeletal muscle atrophy and microvascular rarefaction. Hydrogen sulfide (H2S) has a potential role in skeletal muscle regeneration. However, the mechanisms still need to be elucidated. This is the first study to explore the effect of Sodium hydrosulfide (NaHS) H2S donor, against Dexamethasone (Dex)-induced soleus muscle atrophy and microvascular rarefaction and on muscle endothelial progenitors and M2 macrophages. Rats received either; saline, Dex (0.6 mg/Kg/day), Dex + NaHS (5 mg/Kg/day), or Dex + Aminooxyacetic acid (AOAA), a blocker of H2S (10 mg/Kg/day) for two weeks. The soleus muscle was examined for contractile properties. mRNA expression for Myostatin, Mechano-growth factor (MGF) and NADPH oxidase (NOX4), HE staining, and immunohistochemical staining for caspase-3, CD34 (Endothelial progenitor marker), vascular endothelial growth factor (VEGF), CD31 (endothelial marker), and CD163 (M2 macrophage marker) was performed. NaHS could improve the contractile properties and decrease oxidative stress, muscle atrophy, and the expression of NOX4, caspase-3, Myostatin, VEGF, and CD31 and could increase the capillary density and expression of MGF with a significant increase in expression of CD34 and CD163 as compared to Dex group. However, AOAA worsened the studied parameters. Therefore, H2S can be a promising target to attenuate muscle atrophy and microvascular rarefaction.

Abstract P1035: Co-culturing Human Induced Pluripotent Stem Cell-derived Cardiomyocytes With Mesenchymal Stem Cells Promotes Their Maturation And Enhances Cardioprotection Under Hypoxic Stress

Introduction: Co-culturing human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiCMs) with other stem/progenitor cells like bone-marrow mesenchymal stem cells (BM-MSCs) and endothelial progenitors has been shown to improve their survival post transplantation into ischemic hearts. Co-culturing with other cardiac cells has also shown to improve the maturation of hiCMs in vitro. However, the potential of co-culturing hiPSC-derived MSCs (hiMSCs) for hiCM maturation and cardioprotection has not been explored. Hypothesis: We hypothesize that co-culturing of hiMSCs with hiCMs significantly alters the cells’ proteome, improves hiCM maturation, and enhances cardioprotection. Results: hiCMs were co-cultured with hiMSCs for one week at a ratio of 3:1. To evaluate the effect of hiMSCs on hiCM maturation in the co-culture, changes in the hiCM function and gene expression were assessed via multi electrode array (MEA) analysis and qPCR, respectively. MEA analysis showed an increase in ADP50 and ADP90 in addition to improved response to cardiac drugs in hiCMs co-cultured with hiMSCs as compared to hiCM-only cultures, indicating improved maturation of hiCMs in the co-culture. Furthermore, gene expression analysis showed an increased expression of mature CM-associated genes (MYH7, TNNT2). When subjected to hypoxic stress (1% O 2 ), we also observed a significant decrease in the number of TUNEL positive cells in the co-cultures as compared to hiCM-only culture. Assessment of hiCM functionality via MEA analysis showed an improved cardioprotection in co-cultures as compared to the single cell-type cultures. Furthermore, proteomic analysis demonstrated increased expression of cardioprotective growth factors (GFs): HGF, VEGFA and IGFBP-1 in the conditioned medium of the co-cultured cells versus single cell-type cultures. Conclusion: Overall, our results established that co-culturing hiCMs with hiMSCs improved cardioprotection via increased the expression of cardioprotective growth factors and enhanced the maturation of hiCMs in vitro.

Medicinal signaling cells niche in stromal vascular fraction from lipoaspirate and microfragmented counterpart

AimTo expand our previous findings by increasing the number of patients in a study characterizing medicinal signaling cells (MSC) of stromal vascular fraction from lipoaspirate (SVF-LA) and from microfragmented lipoaspirate (SVF-MLA) applied for the treatment of osteoarthritis (OA).MethodsTwenty OA patients, including 8 new patients, acquiring autologous microfragmented adipose tissue were enrolled. In-parallel immunophenotyping of SVF-LA and SVF-MLA was performed. The samples were incubated in a DuraClone SC prototype tube targeting the CD31, CD34, CD45, CD73, CD90, CD105, and CD146 surface markers, stained with the DRAQ7 cell nuclear dye and Live/Dead Yellow Fixable Stain, and analyzed by flow cytometry.ResultsThe population phenotypes in SVF-LA and SVF-MLA samples included CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells. Compared with the autologous SVF-LA samples, the prevailing cell type in SVF-MLA were EP, which outnumbered leukocytes and supra-adventitial-adipose stromal cells (SA-ASC). The ratio of progenitor cells in SVF-MLA samples differed between female and male patients, showing a higher EP-pericyte and pericyte-SA-ASC ratio in men.ConclusionOur results, hallmarked by EP-enriched anti-inflammatory features and indicating a possible sex-specific impact, contribute to defining the cellular composition of the clinically applied MSC serving as a regenerative cell therapy in OA.

Next generation personalized blood vessel‐on‐chip: Mimicking patient‐specific pathophysiology in sickle cell disease through blood‐derived endothelial progenitors

Advances in tissue engineering and organ‐chip technology have accelerated in vitro research of the vascular system. Incorporating patient‐derived endothelial cells either through direct tissue biopsies or stem cell differentiation techniques further enhance the impact of organ‐chips on precision medicine. However, these methods pose certain challenges and might be difficult to adopt in lab, thereby limiting the predictive power of organ‐chips. Here we report the use of Blood Outgrowth Endothelial Cells (BOECs) directly isolated from patient blood as an alternative to primary and iPSC‐derived endothelial cells for organ‐chip applications. BOECs exhibit the gold‐standard endothelial hallmarks of the “cobblestone” morphology in vitro. When compared to primary HUVECs and iPSC‐derived endothelial cells, healthy BOECs reveal similar levels of growth rates, migration capabilities, de novo vessel formation, response to fluid shear stress and exogenous cytokine inflammation in vitro. To study patient‐specificity, we derive BOECs from two sickle cell disease (SCD) patients with known differences in clinical severity of the disease. Through next generation RNA sequencing, differential gene expression (DGE) analysis and qRT‐PCR, we reveal that SCD patient‐derived BOECs are indeed activated and that the more severe SCD patient exhibited a greater magnitude of activation. This is further confirmed by real‐time recalcified whole blood perfusion through endothelialized vessel‐chips. Blood‐derived endothelial cells may be employed in preclinical research for developing more robust and personalized next‐generation disease models that can ultimately enable clinicians in identifying individuals at high risk of stroke or cardiovascular complications.

Fate mapping and scRNA sequencing reveal origin and diversity of lymph node stromal precursors

Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.

Osteoinductive hybrid hydrogel membranes for in situ bone regeneration in hyperglycemia

In hyperglycemia patients, suffering from insufficient vascularization and vascular network lesion, tissue regeneration, such as bone repair, is limited and maybe delayed by the secondary injury and hyperglycemic microenvironment. Typically, dental therapies involving guided bone regeneration is facing a difficult condition in the patients with diabetes. In this study, a hybrid membrane was endowed with biomimetic function to create an angiogenesis-inductive microenvironment by calcium ion release to overcome the limitations of bone tissue regeneration in diabetic patients. Biomineralized calcium resource was Janus-structured onto the surface of hybrid hydrogel by layer-by-layer technique to enhance vascularization and improve the bone regeneration in this study. The release of calcium ions from mineralized phases was controlled by the solubility of inorganic phases and the degradation of gels, promoting HIF-1α expression and creating a key role in angiogenesis stimulation. With highly enhanced calcium signaling and blood vessel formation, the hybrid hydrogel membranes improved the recruitment, proliferation and differentiation of mesenchymal stem cells and endothelial progenitors, confirmed by the enhancement of microvascular regeneration and new bone formation in the critical-sized calvarial defect in diabetic model in vivo. Our study demonstrates a translational potential of hybrid hydrogels engineered with inorganic minerals for orthopedic applications in hyperglycemia.

Simulated Microgravity Potentiates Hematopoietic Differentiation of Human Pluripotent Stem Cells and Supports Formation of 3D Hematopoietic Cluster.

Microgravity has been shown to induces many changes in proliferation, differentiation and growth behavior of stem cells. Little is known about the effect of microgravity on hematopoietic differentiation of pluripotent stem cells (PSCs). In this study, we used the random position machine (RPM) to investigate whether simulated microgravity (SMG) allows the induction of hematopoietic stem/progenitor cell (HSPC) derived from human embryonic stem cells (hESCs) in vitro. The results showed that SMG facilitates hESCs differentiate to HSPC with more efficient induction of CD34+CD31+ hemogenic endothelium progenitors (HEPs) on day 4 and CD34+CD43+ HSPC on day 7, and these cells shows an increased generation of functional hematopoietic cells in colony-forming unit assay when compared with normal gravity (NG) conditions. Additionally, we found that SMG significantly increased the total number of cells on day 4 and day 7 which formed more 3D cell clusters. Transcriptome analysis of cells identified thousands of differentially expressed genes (DEGs) between NG and SMG. DEGs down-regulated were enriched in the axonogenesis, positive regulation of cell adhesion, cell adhesion molecule and axon guidance, while SMG resulted in the up-regulation of genes were functionally associated with DNA replication, cell cycle, PI3K-Akt signaling pathway and tumorigenesis. Interestingly, some key gene terms were enriched in SMG, like hypoxia and ECM receptor interaction. Moreover, HSPC obtained from SMG culture conditions had a robust ability of proliferation in vitro. The proliferated cells also had the ability to form erythroid, granulocyte and monocyte/macrophage colonies, and can be induced to generate macrophages and megakaryocytes. In summary, our data has shown a potent impact of microgravity on hematopoietic differentiation of hPSCs for the first time and reveals an underlying mechanism for the effect of SMG on hematopoiesis development.

Stem cells mobilization by cardiopulmonary bypass after coronary artery bypass grafting.

Expression of haematopoietic stem and progenitor cells occurs as a result of an inflammatory reaction caused by a damaged organ. Patients undergoing coronary artery bypass surgery (CABG) using cardiopulmonary bypass (CPB) are exposed to an inflammatory reaction, which may be a trigger in the mobilization of regenerative cells. To assess the impact of CPB on stem and progenitor cells mobilization in patients scheduled for CABG. Twenty patients with stable coronary disease, who were scheduled for CABG, were included in the study. Peripheral blood samples were collected perioperatively: 2 h before surgery, before CPB, at CPB weaning, 24 h postoperatively, and on the 6th postoperative day. Analyses of immune-labelled cells were carried out on an ImageStream (IS) system. The following cell populations were identified during cardiac surgery: haematopoietic stem cells (HSC), mesenchymal cells (MSC), endothelial progenitors (EPC), and very small embryonic-like cells (VSEL). The profile of cell mobilization coincides with the perioperative inflammatory response. Mobilization of stem and progenitor cells induced by CPB did not occur in any of the isolated cell lines (p > 0.05). The expression profile of stem and progenitor cells was related with the inflammatory reaction associated with traumatic stress in all cell lines. Type 2 diabetes is a disease that is hampering the trend of MSC mobilization. Mobilization of haematopoietic stem and progenitor cells was not related with CPB. The inflammatory reaction was associated with perioperative trauma. Cell release was inhibited in patients with diabetes.