Primary Progenitor Research Articles

In vivo CRISPR/Cas9 screening identifies Pbrm1 as a regulator of myeloid leukemia development in mice

AbstractCRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin–remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.

Bio-Enhanced Neoligaments Graft Bearing FE002 Primary Progenitor Tenocytes: Allogeneic Tissue Engineering & Surgical Proofs-of-Concept for Hand Ligament Regenerative Medicine.

Hand tendon/ligament structural ruptures (tears, lacerations) often require surgical reconstruction and grafting, for the restauration of finger mechanical functions. Clinical-grade human primary progenitor tenocytes (FE002 cryopreserved progenitor cell source) have been previously proposed for diversified therapeutic uses within allogeneic tissue engineering and regenerative medicine applications. The aim of this study was to establish bioengineering and surgical proofs-of-concept for an artificial graft (Neoligaments Infinity-Lock 3 device) bearing cultured and viable FE002 primary progenitor tenocytes. Technical optimization and in vitro validation work showed that the combined preparations could be rapidly obtained (dynamic cell seeding of 105 cells/cm of scaffold, 7 days of co-culture). The studied standardized transplants presented homogeneous cellular colonization in vitro (cellular alignment/coating along the scaffold fibers) and other critical functional attributes (tendon extracellular matrix component such as collagen I and aggrecan synthesis/deposition along the scaffold fibers). Notably, major safety- and functionality-related parameters/attributes of the FE002 cells/finished combination products were compiled and set forth (telomerase activity, adhesion and biological coating potentials). A two-part human cadaveric study enabled to establish clinical protocols for hand ligament cell-assisted surgery (ligamento-suspension plasty after trapeziectomy, thumb metacarpo-phalangeal ulnar collateral ligamentoplasty). Importantly, the aggregated experimental results clearly confirmed that functional and clinically usable allogeneic cell-scaffold combination products could be rapidly and robustly prepared for bio-enhanced hand ligament reconstruction. Major advantages of the considered bioengineered graft were discussed in light of existing clinical protocols based on autologous tenocyte transplantation. Overall, this study established proofs-of-concept for the translational development of a functional tissue engineering protocol in allogeneic musculoskeletal regenerative medicine, in view of a pilot clinical trial.

Base editors dissect genetic variants in human hematopoietic cells on a large scale

In a recent study, Martin-Rufino and colleagues combined massively parallel base editing in primary human hematopoietic stem and progenitor cells (HSPCs) with functional and single-cell transcriptomic readouts. A series of proof-of-principle experiments highlight the breadth of applications made possible with this approach, which range from gene therapy and immunotherapy, to characterizing single nucleotide variants.

RTK signalling promotes epithelial columnar cell shape and apical junction maintenance in human lung progenitor cells.

Multipotent epithelial progenitor cells can be expanded from human embryonic lungs as organoids and maintained in a self-renewing state using a defined medium. The organoid cells are columnar, resembling the cell morphology of the developing lung tip epithelium in vivo. Cell shape dynamics and fate are tightly coordinated during development. We therefore used the organoid system to identify signalling pathways that maintain the columnar shape of human lung tip progenitors. We found that EGF, FGF7 and FGF10 have distinct functions in lung tip progenitors. FGF7 activates MAPK/ERK and PI3K/AKT signalling, and is sufficient to promote columnar cell shape in primary tip progenitors. Inhibitor experiments show that MAPK/ERK and PI3K/AKT signalling are key downstream pathways, regulating cell proliferation, columnar cell shape and cell junctions. We identified integrin signalling as a key pathway downstream of MAPK/ERK in the tip progenitors; disrupting integrin alters polarity, cell adhesion and tight junction assembly. By contrast, stimulation with FGF10 or EGF alone is not sufficient to maintain organoid columnar cell shape. This study employs organoids to provide insight into the cellular mechanisms regulating human lung development.

Identification of curaxin as a potential new therapeutic for JAK2 V617F mutant patients.

Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms.

Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution.

Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

Anti-tumor efficacy of anti-GD2 CAR NK-92 cells in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are rare and fatal pediatric brainstem gliomas with no cure. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have been proven effective in treating glioblastoma (GBM) in preclinical studies. However, there are no relevant studies on the CAR-NK treatment for DIPG. Our study is the first to evaluate the anti-tumor activity and safety of GD2-CAR NK-92 cells treatment for DIPG. Five patient-derived DIPG cells and primary pontine neural progenitor cell (PPC) were used to access disialoganglioside GD2 expression. Cell killing activity of GD2-CAR NK-92 cells was analyzed by in vitro cytotoxicity assays. Two DIPG patient-derived xenograft models were established to detect the anti-tumor efficacy of GD2-CAR NK-92 cells in vivo. Among the five patient-derived DIPG cells, four had high GD2 expression, and one had low GD2 expression. In in vitro assays, GD2-CAR NK-92 cells could effectively kill DIPG cells with high GD2 expression while having limited activity against DIPG cells with low GD2 expression. In in vivo assays, GD2-CAR NK-92 cells could inhibit tumor growth in TT150630 DIPG patient-derived xenograft mice (high GD2 expression) and prolong the overall survival of the mice. However, GD2-CAR NK-92 showed limited anti-tumor activity for TT190326DIPG patient-derived xenograft mice (low GD2 expression). Our study demonstrates the potential and safety of GD2-CAR NK-92 cells for adoptive immunotherapy of DIPG. The safety and anti-tumor effect of this therapy need to be further demonstrated in future clinical trials.

Paraquat-induced neurogenesis abnormalities via Drp1-mediated mitochondrial fission

Neurogenesis is a fundamental process in the development and plasticity of the nervous system, and its regulation is tightly linked to mitochondrial dynamics. Imbalanced mitochondrial dynamics can result in oxidative stress, which has been implicated in various neurological disorders. Paraquat (PQ), a commonly used agricultural chemical known to be neurotoxic, induces oxidative stress that can lead to mitochondrial fragmentation. In this study, we investigated the effects of PQ on neurogenesis in primary murine neural progenitor cells (mNPCs) isolated from neonatal C57BL/6 mice. We treated the mNPCs with 0–40 μM PQ for 24 h and observed that PQ inhibited their proliferation, migration, and differentiation into neurons in a concentration-dependent manner. Moreover, PQ induced excessive mitochondrial fragmentation and upregulated the expression of Drp-1, p-Drp1, and Fis-1, while downregulating the expression of Mfn2 and Opa1. To confirm our findings, we used Mdivi-1, an inhibitor of mitochondrial fission, which reversed the adverse effects of PQ on neurogenesis, particularly differentiation into neurons and migration of mNPCs. Additionally, we found that Mito-TEMPO, a mitochondria-targeted antioxidant, ameliorated excessive mitochondrial fragmentation caused by PQ. Our study suggests that PQ exposure impairs neurogenesis by inducing excessive mitochondrial fission and abnormal mitochondrial fragmentation via oxidative stress. These findings identify mitochondrial fission as a potential therapeutic target for PQ-induced neurotoxicity. Further research is needed to elucidate the underlying mechanisms of mitochondrial dynamics and neurogenesis in the context of oxidative stress-induced neurological disorders.

Distinct human skeletal muscle-derived CD90 progenitor subsets for myo-fibro-adipogenic disease modeling and treatment in multiplexed conditions.

Chronic muscle injuries, such as massive rotator cuff tears, are associated with progressive muscle wasting, fibrotic scarring, and intramuscular fat accumulation. While progenitor cell subsets are usually studied in culture conditions that drive either myogenic, fibrogenic, or adipogenic differentiation, it is still unknown how combined myo-fibro-adipogenic signals, which are expected to occur in vivo, modulate progenitor differentiation. We therefore evaluated the differentiation potential of retrospectively generated subsets of primary human muscle mesenchymal progenitors in multiplexed conditions in the presence or absence of 423F drug, a modulator of gp130 signaling. We identified a novel CD90+CD56- non-adipogenic progenitor subset that maintained a lack of adipogenic potential in single and multiplexed myo-fibro-adipogenic culture conditions. CD90-CD56- demarcated fibro-adipogenic progenitors (FAP) and CD56+CD90+ progenitors were typified as myogenic. These human muscle subsets exhibited varying degrees of intrinsically regulated differentiation in single and mixed induction cultures. Modulation of gp130 signaling via 423F drug mediated muscle progenitor differentiation in a dose-, induction-, and cell subset-dependent manner and markedly decreased fibro-adipogenesis of CD90-CD56- FAP. Conversely, 423F promoted myogenesis of CD56+CD90+ myogenic subset, indicated by increased myotube diameter and number of nuclei per myotube. 423F treatment eliminated FAP-derived mature adipocytes from mixed adipocytes-FAP cultures but did not modify the growth of non-differentiated FAP in these cultures. Collectively, these data demonstrate that capability of myogenic, fibrogenic, or adipogenic differentiation is largely dependent on the intrinsic features of cultured subsets, and that the degree of lineage differentiation varies when signals are multiplexed. Moreover, our tests performed in primary human muscle cultures reveal and confirm the potential triple-therapeutic effects of 423F drug which simultaneously attenuates degenerative fibrosis, fat accumulation and promotes myo-regeneration.

Ciliary neurotrophic factor stimulates stem/progenitor Leydig cell proliferation but inhibits differentiation into its lineage in rats.

Ciliary neurotrophic factor (CNTF) is a member of the interleukin-6 family of cytokines. CNTF drives many cells for their development. However, its effects on Leydig cells (LCs) development remains unclear. In the current study, we used 3D seminiferous tubule culture system to induce the proliferation and differentiation of tubule-associated stem Leydig cells (SLCs) and primary progenitor Leydig cells (PLCs) culture to address the effects of CNTF. We found that CNTF stimulated the proliferation of SLCs but inhibited its development to the LCs lineage. The CNTF-mediated effects can be reversed by STAT3 inhibitor S3I-201 and PI3K inhibitor wortmannin, indicating that CNTF acts via STAT3-PI3K signaling pathways to increase stem/progenitor Leydig cell proliferation. CNTF at 1 and 10 ng/mL significantly decreased androgen production by PLCs. Microarray analysis for CNTF-treated PLCs showed that CNTF blocked steroidogenic pathways through downregulating Scarb1, Star and Hsd3b1 possibly through the downregulating the transcription factor Nr5a1 expression. CNTF stimulates the proliferation but blocks the differentiation of stem/progenitor Leydig cells. This article is protected by copyright. All rights reserved.

Primary Progenitor Tenocytes: Cytotherapeutics and Cell-Free Derivatives

Primary progenitor tenocytes are diploid cells that may be cultured in vitro and therapeutically used for allogeneic musculoskeletal regenerative medicine. Firstly, technical aspects of cell banking, biotechnological manufacturing, and extensive preclinical characterization data have confirmed that FE002-Ten primary progenitor tenocytes may be safely considered for human cytotherapeutic use (e.g., in tissue engineering products, standardized transplants). Parallelly, lyophilized progenitor tenocyte extracts (e.g., stabilized cells or cell-free derivatives) were shown to optimally act as potent hyaluronan-based hydrogel functionalizing agents, useful for stability enhancement against oxidative product degradation. Therefore, primary progenitor tenocytes (e.g., FE002-Ten cell source) may potentially be used in diverse clinical presentations of tendon-related pathologies, ranging from volumetric tissue replacement (i.e., for the promotion of enhanced graft bio-integration) to local management of tissular inflammation and pain (i.e., ancillary action of the cellular extracts for the functional enhancement of injectable hyaluronan-based preparations). Overall, the primary progenitor tenocytes investigated under the Swiss progenitor cell transplantation program were shown to represent highly standardized biotechnological materials with a versatility of potential therapeutic uses after formulation into an array of cytotherapeutic preparations or cell-free devices.

Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

Upregulated estrogen receptors impairs myogenesis and elevates adipogenesis related factor levels in the paravertebral muscles of patients with idiopathic scoliosis

The prevalence of idiopathic scoliosis (IS) is 2–3% worldwide and is more common in girls. Estrogen receptors (ERs) is supposed to be related to sex differences and development of IS. Meanwhile, paravertebral muscle (PVM) abnormalities play important roles in the pathogenesis of IS. But the changes of ERs between the PVMs from IS patients and controls, and the mechanism by which ERs may affect IS patients remain unclear. Thus, the expression levels of ERs, myogenesis regulator (MYOG) and adipogenesis related factors (CEBPA, PPARγ, FABP4), as well as morphological changes in the PVMs and primary skeletal muscle mesenchymal progenitor cells (hSM-MPCs) of IS patients and controls were investigated. Increased expression levels of ERs and CEBPA, PPARγ, FABP4, together with severe myofiber necrosis and fat infiltration, were found in the PVMs of IS patients. Meanwhile, upregulated ERs, FABP4 and CEBPA, downregulated MYOG and impaired myogenesis were also revealed in the hSM-MPCs of IS patients compared with those of controls. Upregulation of ERs inhibited myogenesis but increased expression of CEBPA and FABP4 in C2C12 myoblasts. Nevertheless, treatment of ER antagonist increased expression of MYOG, enhanced myogenesis and decreased expression of CEBPA and FABP4 in skeletal muscle cells of IS patients. Therefore, our study suggested that PVMs specific upregulation of ERs could impair myogenesis and increase the expression of adipogenesis related factors, further leading to PVMs abnormalities in IS patients.

Human cytomegalovirus pUL97 upregulates SOCS3 expression via transcription factor RFX7 in neural progenitor cells

Congenital human cytomegalovirus (HCMV) infection causes severe damage to the fetal brain, and the underlying mechanisms remain elusive. Cytokine signaling is delicately controlled in the fetal central nervous system to ensure proper development. Here we show that suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the IL-6 cytokine family signaling, was upregulated during HCMV infection in primary neural progenitor cells (NPCs) with a biphasic expression pattern. From viral protein screening, pUL97 emerged as the viral factor responsible for prolonged SOCS3 upregulation. Further, by proteomic analysis of the pUL97-interacting host proteins, regulatory factor X 7 (RFX7) was identified as the transcription factor responsible for the regulation. Depletion of either pUL97 or RFX7 prevented the HCMV-induced SOCS3 upregulation in NPCs. With a promoter-luciferase activity assay, we demonstrated that the pUL97 kinase activity and RFX7 were required for SOCS3 upregulation. Moreover, the RFX7 phosphorylation level was increased by either UL97-expressing or HCMV-infection in NPCs, suggesting that pUL97 induces RFX7 phosphorylation to drive SOCS3 transcription. We further revealed that elevated SOCS3 expression impaired NPC proliferation and migration in vitro and caused NPCs migration defects in vivo. Taken together, these findings uncover a novel regulatory mechanism of sustained SOCS3 expression in HCMV-infected NPCs, which perturbs IL-6 cytokine family signaling, leads to NPCs proliferation and migration defects, and consequently affects fetal brain development.

Ethics & exclusions: Reflections on the passing of Stephen Castles

Ethics & exclusions: Reflections on the passing of Stephen Castles

MicroRNA-eQTLs in the developing human neocortex link miR-4707-3p expression to brain size.

Expression quantitative trait loci (eQTL) data have proven important for linking non-coding loci to protein-coding genes. But eQTL studies rarely measure microRNAs (miRNAs), small non-coding RNAs known to play a role in human brain development and neurogenesis. Here, we performed small-RNA sequencing across 212 mid-gestation human neocortical tissue samples, measured 907 expressed miRNAs, discovering 111 of which were novel, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and brain size phenotypes, where the miRNA expression increasing allele was associated with decreased brain size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells decreased expression of predicted targets and increased cell proliferation, indicating miR-4707-3p modulates progenitor gene regulation and cell fate decisions. Integrating miRNA-eQTLs with existing GWAS yielded evidence of a miRNA that may influence human brain size and function via modulation of neocortical brain development.

Lyophilized Progenitor Tenocyte Extracts: Sterilizable Cytotherapeutic Derivatives with Antioxidant Properties and Hyaluronan Hydrogel Functionalization Effects.

Cultured primary progenitor tenocytes in lyophilized form were previously shown to possess intrinsic antioxidant properties and hyaluronan-based hydrogel viscosity-modulating effects in vitro. The aim of this study was to prepare and functionally characterize several stabilized (lyophilized) cell-free progenitor tenocyte extracts for inclusion in cytotherapy-inspired complex injectable preparations. Fractionation and sterilization methods were included in specific biotechnological manufacturing workflows of such extracts. Comparative and functional-oriented characterizations of the various extracts were performed using several orthogonal descriptive, colorimetric, rheological, mechanical, and proteomic readouts. Specifically, an optimal sugar-based (saccharose/dextran) excipient formula was retained to produce sterilizable cytotherapeutic derivatives with appropriate functions. It was shown that extracts containing soluble cell-derived fractions possessed conserved and significant antioxidant properties (TEAC) compared to the freshly harvested cellular starting materials. Progenitor tenocyte extracts submitted to sub-micron filtration (0.22 µm) and 60Co gamma irradiation terminal sterilization (5-50 kGy) were shown to retain significant antioxidant properties and hyaluronan-based hydrogel viscosity modulating effects. Hydrogel combination products displayed important efficacy-related characteristics (friction modulation, tendon bioadhesivity) with significant (p < 0.05) protective effects of the cellular extracts in oxidative environments. Overall, the present study sets forth robust control methodologies (antioxidant assays, H2O2-challenged rheological setups) for stabilized cell-free progenitor tenocyte extracts. Importantly, it was shown that highly sensitive phases of cytotherapeutic derivative manufacturing process development (purification, terminal sterilization) allowed for the conservation of critical biological extract attributes.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

CUT & RUN to Profile Chromatin-Bound Proteins in Primary Mouse Neural Progenitor Cells.

Cleavage under targets and release using nuclease (CUT & RUN) is an innovative method to profile histone modifications and chromatin-bound proteins genome-wide. CUT & RUN offers two distinct advantages of requiring much fewer cells and providing strong signal-to-noise ratios in deep-sequencing data. Here, we describe a workflow starting from dissociation and sorting of mouse embryonic brains, CUT & RUN, and DNA library preparation to deep sequencing. With our workflow, researchers can obtain high-quality sequencing data to profile histones and chromatin-associated proteins by using as few as 100,000 neural progenitor cells (NPCs).