Enrichment Of Stem Cells Research Articles

Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

Cancer stem cell (CSC) is a challenge in the therapy of triple-negative breast cancer (TNBC). Intratumoral hypoxia is a common feature of solid tumor. Hypoxia may contribute to the maintenance of CSC, resulting in a poor efficacy of traditional treatment and recurrence of TNBC cases. However, the underlying molecular mechanism involved in hypoxia-induced CSC stemness maintenance remains unclear. Here, we report that hypoxia stimulated DNA double-strand breaks independent of ATM kinase activation (called oxidized ATM in this paper) play a crucial role in TNBC mammosphere formation and stemness maintenance by governing a specific energy metabolism reprogramming (EMR). Oxidized ATM up-regulates GLUT1, PKM2, and PDHa expressions to enhance the uptake of glucose and production of pyruvate rather than lactate products, which facilitates glycolytic flux to mitochondrial pyruvate and citrate, thus resulting in accumulation of cytoplasmic acetyl-CoA instead of the tricarboxylic acid (TCA) cycle by regulating ATP-citrate lyase (ACLY) activity. Our findings unravel a novel model of TNBC-CSC glucose metabolism and its functional role in maintenance of hypoxic TNBC-CSC stemness. This work may help us to develop new therapeutic strategies for TNBC treatment.

Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno‐magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF‐p75‐SALL2 signaling pathway was involved in regulating the differentiation of CjECs.

Additional Evidence to Establish Existence of Two Stem Cell Populations Including VSELs and SSCs in Adult Mouse Testes.

Present study aims to describe a simple and robust protocol to delineate the presence of pluripotent, very small embryonic-like stem cells (VSELs) in addition to spermatogonial stem cells (SSCs) in adult mouse testes. Testicular seminiferous tubules were subjected to enzymatic dissociation to obtain single cells suspension. Stem cells were enriched by spinning at different speeds wherein majority of somatic cells were pelleted at 1000rpm (250g, Pellet A) and putative stem cells by spinning the supernatant (obtained after separating Pellet A) at 3000rpm (1000g, Pellet B). Viable (7AAD-ve), 2-6μm, LIN-CD45-SCA-1+ VSELs were studied after doublets exclusion by flow cytometry in both Pellets A & B. Almost ten-fold enrichment of VSELs was obtained in Pellet B (0.27+0.05%) compared to Pellet A (0.03+0.003%). SCA-1 expressing SSCs (>6μm, 0.18+0.06%) were clearly distinguished from VSELs (2-6μm, 0.07+0.003%) by flow cytometry studies on total testicular cells suspension collected by spinning at 3000rpm. Enriched stem cells from Pellet B were used to study expression of OCT-4, NANOG, SCA-1, SSEA-1, LIFR, GFRa, c-KIT, ERα and ERβ. Cells in Pellet B were also subjected to RT-PCR to study pluripotent (Oct-4a, Sox2, Nanog), primordial germ cells (Stella, Fragilis), SSCs (Oct-4) and estrogen receptors (ERα and ERβ) specific transcripts. qRT-PCR analysis showed >2 folds up-regulation of stem cell markers in Pellet B (Oct-4A, Oct-4, Sox2, Nanog) compared to Pellet A. To conclude, spinning at higher speed led to successful enrichment of pluripotent VSELs from testes which have remained ignored till now. Expression of ERα & β on VSELs/SSCs makes them vulnerable to endocrine disruption.

MicroRNA Profiling of Highly Enriched Human Corneal Epithelial Stem Cells by Small RNA Sequencing

The objective of the study was to elucidate the microRNA (miRNA) profile of an enriched human corneal epithelial stem cell (CESC) population in comparison to differentiated central corneal epithelial cells (CCECs) by small RNA sequencing. The CESCs were enriched by differential enzymatic treatment to isolate the basal limbal epithelial cells followed by laser capture microdissection of cells with nucleus to cytoplasm ratio ≥0.7, from donor tissues. Small RNA sequencing was carried out using Illumina NextSeq. 500 platform and the validation of differentially expressed miRNAs by quantitative real-time PCR (qPCR) and locked nucleic acid miRNA in-situ hybridization (LNA-ISH). The sequencing identified 62 miRNAs in CESCs and 611 in CCECs. Six miRNAs: hsa-miR-21-5p, 3168, 143-3p, 10a-5p, 150-5p and 1910-5p were found to be significantly upregulated in enriched CESCs, which was further confirmed by qPCR and LNA-ISH. The expression of hsa-miR-143-3p was exclusive to clusters of limbal basal epithelial cells. The targets of the upregulated miRNAs were predicted to be associated with signaling pathways -Wnt, PI3K-AKT, MAPK and pathways that regulate pluripotency of stem cells, cell migration, growth and proliferation. Further studies are essential to elucidate their functional role in maintenance of stemness. The findings of the study also hypothesize the inherent potential of hsa-miR-143-3p to serve as a biomarker for identifying CESCs.

Characterization of proliferation, differentiation potential, and gene expression among clonal cultures of human dental pulp cells

Mesenchymal stem cells are a highly promising source of cells for regeneration therapy because of their multilineage differentiation potential. However, distinct markers for mesenchymal stem cells are not well-established. To identify new candidate marker genes for multipotent human dental pulp stem cells, we analyzed the characteristics and gene expression profiles of cell clones obtained from a single dental pulp specimen derived from an 11-year-old female patient. Fifty colony-forming single cell-derived clones were separately cultured until the cessation of growth. These clones varied in their proliferation abilities and surface marker (STRO-1 and CD146) expression patterns, as well as their odontogenic, adipogenic, and chondrogenic differentiation potentials. Four clones maintained their original differentiation potentials during long-term culture. Gene expression profile by DNA microarray analysis of five representative clones identified 1227 genes that were related to multipotency. Ninety of these 1227 genes overlapped with genes reportedly involved in ‘stemness or differentiation’. Based on the predicted locations of expressed protein products and large changes in expression levels, 14 of the 90 genes were selected as candidate dental pulp stem cell markers, particularly in relation to their multipotency characteristics. This characterization of cell clones obtained from a single specimen of human dental pulp provided information regarding new candidate marker genes for multipotent dental pulp stem cells, which could facilitate efficient analysis or enrichment of multipotent stem cells.

Recent advances in targeting cancer stem cells using oncolytic viruses.

Oncolytic virotherapy is a promising antitumor strategy which utilizes the lytic nature of viral replication to kill cancer cells. Oncolytic viruses (OVs) can induce cancer cell death and trigger immune responses to metastatic cancer in vivo. Reverse genetic systems have aided the insertion of anticancer genes into various OVs to augment their oncolytic capacity. Furthermore, OVs target and destroy the population of tumor-initiating cancer stem cells. These cancer stem cells are associated with metastasis and development of resistance to conventional anticancer approaches. Targeting cancer stem cells is essential since killing only differentiated tumor cells may lead to enrichment of cancer stem cells and thus indicate a poor prognosis. In this review, we summarize the oncolytic activity of various classes of OVs towards different types of cancer stem cells and also discuss the synergistic activity achieved by the combination of OVs with traditional therapies on chemo- and radiotherapy-resistant cancer stem cells.

Cancer stem cell enrichment is associated with enhancement of nicotinamide N-methyltransferase expression.

The cancer stem cell theory states that a subset of tumor cells, termed cancer stem cells (CSCs), has the ability to self-renew and differentiate within the tumors. According to this theory, CSCs would be mainly responsible for tumor initiation, progression, resistance to therapy, recurrence, and metastasis. In this study, a culture system was setup to enrich CSCs from bladder cancer (T24), lung cancer (A549), colorectal cancer (CaCo-2), and osteosarcoma (MG63) cell lines, through sphere formation. Magnetic-activated cell sorting was also used to further increase CSC enrichment. Subsequently, molecular characterization of CSC-enriched cell populations and parental cells was carried out, by exploring the expression levels of stem markers and the enzyme nicotinamide N-methyltransferase (NNMT). Results obtained showed a significant upregulation of stem cell markers in CSC-enriched populations, obtained upon sphere formation, compared with parental counterparts. Moreover, NNMT expression levels were markedly increased in samples enriched with CSCs with respect to control cells. Considering the fundamental role played by CSCs in carcinogenesis, reported data strengthen the hypothesis that sustains a pivotal role of NNMT in cancer growth and metastasis. In addition, these findings could represent an important achievement for the development of new and effective anticancer therapies, based on CSC-associated targets.

Assessment of Enrichment of Human Mesenchymal Stem Cells Based on Plasma and Mitochondrial Membrane Potentials.

Background: Human mesenchymal stem cells (hMSCs) are utilized preclinically and clinically as a candidate cell therapy for a wide range of inflammatory and degenerative diseases. Despite promising results in early clinical trials, consistent outcomes with hMSC-based therapies have proven elusive in many of these applications. In this work, we attempt to address this limitation through the design of a stem cell therapy to enrich hMSCs for desired electrical and ionic properties with enhanced stemness and immunomodulatory/regenerative capacity. Materials and Methods: In this study, we sought to develop initial protocols to achieve electrically enriched hMSCs (EE-hMSCs) with distinct electrical states and assess the potential relationship with respect to hMSC state and function. We sorted hMSCs based on fluorescence intensity of tetramethylrhodamine ethyl ester (TMRE) and investigated phenotypic differences between the sorted populations. Results: Subpopulations of EE-hMSCs exhibit differential expression of genes associated with senescence, stemness, immunomodulation, and autophagy. EE-hMSCs with low levels of TMRE, indicative of depolarized membrane potential, have reduced mRNA expression of senescence-associated markers, and increased mRNA expression of autophagy and immunomodulatory markers relative to EE-hMSCs with high levels of TMRE (hyperpolarized). Conclusions : This work suggests that the utilization of EE-hMSCs may provide a novel strategy for cell therapies, enabling live cell enrichment for distinct phenotypes that can be exploited for different therapeutic outcomes.

Fat Graft Enrichment Strategies: A Systematic Review.

Autologous fat grafting is a dynamic modality used in plastic surgery as an adjunct to improve functional and aesthetic form. However, current practices in fat grafting for soft-tissue augmentation are plagued by tremendous variability in long-term graft retention, resulting in suboptimal outcomes and repetitive procedures. This systematic review identifies and critically appraises the evidence for various enrichment strategies that can be used to augment and improve the viability of fat grafts. A comprehensive literature search of the Medline and PubMed databases was conducted for animal and human studies published through October of 2017 with multiple search terms related to adipose graft enrichment agents encompassing growth factors, platelet-rich plasma, adipose-derived and bone marrow stem cells, gene therapy, tissue engineering, and other strategies. Data on level of evidence, techniques, complications, and outcomes were collected. A total of 1382 articles were identified, of which 147 met inclusion criteria. The majority of enrichment strategies demonstrated positive benefit for fat graft survival, particularly with growth factors and adipose-derived stem cell enrichment. Platelet-rich plasma and adipose-derived stem cells had the strongest evidence to support efficacy in human studies and may demonstrate a dose-dependent effect. Improved understanding of enrichment strategies contributing to fat graft survival can help to optimize safety and outcomes. Controlled clinical studies are lacking, and future studies should examine factors influencing graft survival through controlled clinical trials in order to establish safety and to obtain consistent outcomes.

Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by motor neuron loss, resulting in muscle wasting, paralysis and eventual death. A key pathological feature of ALS is cytoplasmically mislocalized and aggregated TDP-43 protein in >95% of cases, which is considered to have prion-like properties. Historical studies have predominantly focused on genetic forms of ALS, which represent ∼10% of cases, leaving the remaining 90% of sporadic ALS relatively understudied. Additionally, the role of astrocytes in ALS and their relationship with TDP-43 pathology is also not currently well understood. We have therefore used highly enriched human induced pluripotent stem cell (iPSC)-derived motor neurons and astrocytes to model early cell type-specific features of sporadic ALS. We first demonstrate seeded aggregation of TDP-43 by exposing human iPSC-derived motor neurons to serially passaged sporadic ALS post-mortem tissue (spALS) extracts. Next, we show that human iPSC-derived motor neurons are more vulnerable to TDP-43 aggregation and toxicity compared with their astrocyte counterparts. We demonstrate that these TDP-43 aggregates can more readily propagate from motor neurons into astrocytes in co-culture paradigms. We next found that astrocytes are neuroprotective to seeded aggregation within motor neurons by reducing (mislocalized) cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell-type specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS. In summary, we have developed a human, clinically relevant, and cell-type specific modelling platform that recapitulates key aspects of sporadic ALS and uncovers both an initial neuroprotective role for astrocytes and the cell type-specific toxic effect of TDP-43 oligomers.

Sustained Donor Chimerism and Rapid Immune Cell Reconstitution with a Low Probability of Gvhd Following Familial Haploidentical (FHI) CD34 Enriched Stem Cell Transplantation with Pbmnc Addback in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Background Allogeneic stem cell transplantation (AlloSCT) from an HLA-matched sibling donor is the only known curative therapy in high-risk SCD patients (Talano/Cairo, EJH, 2015). Unfortunately only about 15% of high-risk SCD patients have an HLA-matched unaffected sibling donor. T cell depletion has been employed to reduce AGVHD. We reported a very low incidence of GVHD in pediatric recipients receiving CD34 enriched HPC products with PBMNC addback from MUD donors (Geyer/Cairo et al, BJH, 2012). Rapid NK cell reconstitution after AlloSCT is associated with a significant improvement in 1yr OS (Pical-Izard, BBMT, 2015). Recently, we reported promising results for high-risk SCD patients at 1 yr follow-up after FHI CD34 enriched/PBMNC with addback with the probability of 1-yr OS 88.2% (Cairo, et al, JAMA Peds, 2019). Objective To investigate donor chimerism, immune reconstitution and the incidence of GVHD in high-risk SCD patients following AlloSCT using FHI CD34 enrichment/PBMNC (2 × 105 CD3/kg) addback. Methods CD34 cells were enriched using the CliniMACS® system, kindly provided by Miltenyi Biotec, with a target dose of 10 × 106 CD34+ cells/kg. PBMNC were added back to the final CD34 product at a dose of 2 × 10*5 CD3/kg. Whole blood and CD71-enriched erythroid lineage chimerism were determined by STR. Immune cell, subset reconstitution, NK function, CD107a and granzyme B were assessed by flow cytometry as previously described (Geyer/Cairo et al. BJH, 2012, Chu/Cairo et al, Can Imm Res, 2015). Results The cumulative incidence of grade II-IV and late AGVHD was 6.2 % and moderate and/or severe CGVHD incidence was 6.7%, respectively (Fig. 1A). There was 100% engraftment of neutrophils and platelets. The median day post-HISCT to neutrophil and platelet engraftment was +9 and +19, respectively. Whole blood donor chimerism (mean±SEM) at 1-yr, 2-ys, and 3-ys post-HISCT was 97±1%, 97±1%, 97±1%, respectively (Fig.1B). Donor chimerism for CD71-enriched erythroid lineage cells (mean±SEM) at 1-yr, 2-yrs, 3-yrs post-HISCT was 97±2%, 98±1%, 98±1%, respectively (Fig.1B). The time to recovery of minimal normal levels for CD3 (800 cells/ul), CD4 (400 cells/ul), CD8 (200 cells/ul), & CD19 cells (200 cells/ul), was approximately 365, 365, 270, and 60 days post-HISCT, respectively (Fig.1C). NK reconstitution was rapid and peaked at d+30 (36±9%) with higher level of activating receptors NKp46, NKG2D & KIR2DS and inhibitory receptors NKG2A, CD94 and KIR2DL2/3. NK cytotoxicity against K562 (E:T = 10:1) peaked at d+30 and d+180 vs pre-transplant (p Conclusion The PBMNC addback facilitated rapid donor chimerism & immune reconstitution with a low probability of Grade II-IV AGVHD. The rapid NK reconstitution may have in part contributed to the excellent 1yr OS in this study (FDA R01FD004090 (MSC)). This approach is comparable to the method of CD3 TCRa/b-CD19 depletion.

Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL-associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in supraphysiological levels of signaling activity and are rarely found in human T-ALL. The current protocol describes the method for mouse bone marrow isolation, hematopoietic stem and progenitor cell (HSC) enrichment, followed by retroviral transduction with an oncogenic mutant form of the NOTCH1 receptor (NOTCH1-L1601P-ΔP) that closely resembles the gain-of-function mutations most commonly found in patient samples. A hallmark of this forced expression of constitutively active NOTCH1 is a transient wave of extrathymic immature T-cell development, which precedes oncogenic transformation to T-ALL. Furthermore, this approach models leukemic transformation and progression in vivo by allowing for crosstalk between leukemia cells and the microenvironment, an aspect unaccounted for in cell-line based in vitro studies. Thus, the HSC transduction and transplantation model more faithfully recapitulates development of the human disease, providing a highly comprehensive and versatile tool for further in vivo and ex vivo functional studies.

HGF/c-Met Promote Renal Carcinoma Cancer Stem Cells Enrichment Through Upregulation of Cir-CCDC66.

Increasing studies have suggested that circular RNAs play an important function in the process of numerous cancers. We aimed to investigate the possible role of cir-CCDC66 in renal carcinoma cancer. As cancer stem cells are responsible for the renal carcinoma cancer tumor growth and resistance to conventional therapy, we focus on the cir-CCDC66 influence on renal carcinoma cancer stem cells. In this study, we performed experiments in human renal tubular epithelial cell HK2 cells and several renal carcinoma cancer cancer cell lines. The results showed that cir-CCDC66 was upregulated not only in renal carcinoma cancer cancer cell lines but also in cancer stem cell spheres. What’s more, the results showed that cir-CCDC66 enhanced the cancer stem cell enrichment. Further mechanistic studies showed that hepatocyte growth factor/c-Met pathway was activated in cancer stem cell enrichment and responsible for the cir-CCDC66 upregulation. Inhibition of hepatocyte growth factor/c-Met could block cir-CCDC66-induced cancer stem cell enrichment. In conclusion, our research revealed a novel mechanism between hepatocyte growth factor/c-Met/cir-CCDC66 and cancer stem cell enrichment. We verified that cir-CCDC66 could be a promising biomarker and therapy target for renal carcinoma cancer treatment.

Label-free enrichment of fate-biased human neural stem and progenitor cells

Human neural stem and progenitor cells (hNSPCs) have therapeutic potential to treat neural diseases and injuries since they provide neuroprotection and differentiate into astrocytes, neurons, and oligodendrocytes. However, cultures of hNSPCs are heterogeneous, containing cells linked to distinct differentiated cell fates. HNSPCs that differentiate into astrocytes are of interest for specific neurological diseases, creating a need for approaches that can detect and isolate these cells. Astrocyte-biased hNSPCs differ from other cell types in electrophysiological properties, namely membrane capacitance, and we hypothesized that this could be used to enrich these cells using dielectrophoresis (DEP). We implemented a two-step DEP sorting scheme, consisting of analysis to define the optimal sorting frequency followed by separation of cells at that frequency, to test whether astrocyte-biased cells could be separated from the other cell types present in hNSPC cultures. We developed a novel device that increased sorting reproducibility and provided both enriched and depleted cell populations in a single sort. Astrocyte-biased cells were successfully enriched from hNSPC cultures by DEP sorting, making this the first study to use electrophysiological properties for label-free enrichment of human astrocyte-biased cells. Enriched astrocyte-biased human cells enable future experiments to determine the specific properties of these important cells and test their therapeutic efficacy in animal models of neurological diseases.

Mechanoresponse of stem cells for vascular repair.

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.

Targeting histone deacetylase SIRT1 selectively eradicates EGFR TKI-resistant cancer stem cells via regulation of mitochondrial oxidative phosphorylation in lung adenocarcinoma

Lung adenocarcinoma (LAD) is a human malignancy successfully treated with the tyrosine kinase inhibitor (TKI) gefitinib; however, the enrichment of therapy resistant cancer stem cells (CSCs) in such patients is assumed to be a source of treatment failure. Evaluation of LAD cell populations treated with the TKI inhibitor gefitinib identified unique aspects of a subpopulation of tumor cells exhibiting stem-like properties and mitochondria-specific metabolic features along with their reliance on sirtuin 1 (SIRT1) for survival advantage. This addiction to bioenergetic metabolism in LAD treated with EGFR-targeted therapy suggests that mitochondrial targeting should be synthetically lethal using established cytotoxic therapies. Accordingly, loss of the phenotype present in resistant CSC clones either by targeting the energy metabolism with tigecycline, a mitochondrial DNA-translation inhibitor, or tenovin-6 (TV-6), a SIRT1 inhibitor, inhibited their dependency on mitochondrial oxidative phosphorylation (mtOXPHOS) and sensitized them for a more pronounced and long-lasting TKI therapeutic effect. The results specifically demonstrated that combined therapy with TV-6 and gefitinib resulted in tumor regression in xenograft mouse models, whereas administration of a single agent showed no such efficacy. Importantly, combined treatment with TV-6 also decreased the effective dose of gefitinib necessary for treatment response. Clinical analysis demonstrated that high-profile SIRT1 and mtOXPHOS proteins were associated with recurrence and poor prognosis in LAD patients. These observations support the CSC hypothesis for cancer relapse and advocate use of mitochondria-targeting inhibitors as part of combinatorial therapy in a variety of clinical settings, as well as for reducing first-line TKI dosage in LAD patients.

Comparison and characterization of enriched mesenchymal stem cells obtained by the repeated filtration of autologous bone marrow through porous biomaterials

BackgroundWhen bone marrow is repeatedly filtered through porous material, the mesenchymal stem cells (MSCs) in the bone marrow can adhere to the outer and inner walls of the carrier material to become enriched locally, and this is a promising method for MSC enrichment. In this process, the enrichment efficiency of MSCs involved in the regulation of the cell ecology of postfiltration composites containing other bone marrow components is affected by many factors. This study compared the enrichment efficiency and characterized the phenotypes of enriched MSCs obtained by the filtration of autologous bone marrow through different porous bone substitutes.MethodsHuman bone marrow was filtered through representative porous materials, and different factors affecting MSC enrichment efficiency were evaluated. The soluble proteins and MSC phenotypes in the bone marrow before and after filtration were also compared.ResultsThe enrichment efficiency of the MSCs found in gelatin sponges was 96.1% ± 3.4%, which was higher than that of MSCs found in allogeneic bone (72.5% ± 7.6%) and porous β-TCP particles (61.4% ± 5.4%). A filtration frequency of 5–6 and a bone marrow/material volume ratio of 2 achieved the best enrichment efficiency for MSCs. A high-throughput antibody microarray indicated that the soluble proteins were mostly filtered out and remained in the flow through fluid, whereas a small number of proteins were abundantly (> 50%) enriched in the biomaterial. In terms of the phenotypic characteristics of the MSCs, including the cell aspect ratio, osteogenetic fate, specific antigens, gene expression profile, cell cycle stage, and apoptosis rate, no significant changes were found before or after filtration.ConclusionWhen autologous bone marrow is rapidly filtered through porous bone substitutes, the optimal enrichment efficiency of MSCs can be attained by the rational selection of the type of carrier material, the bone marrow/carrier material volume ratio, and the filtration frequency. The enrichment of bone marrow MSCs occurs during filtration, during which the soluble proteins in the bone marrow are also absorbed to a certain extent. This filtration enrichment technique does not affect the phenotype of the MSCs and thus may provide a safe alternative method for MSC enrichment.

Cancer stem cells enrichment with surface markers CD271 and CD44 in human head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate of 50%. One potential reason for treatment failure is the presence of cancer stem cells (CSCs). Several cell markers, particularly CD44, have been used to isolate CSCs. However, isolating a pure population of CSC in HNSCC still remains a challenging task. Recent findings show that normal oral stem cells were isolated using CD271 as a marker. Thus, we investigated the combined use of CD271 and CD44 to isolate an enriched subpopulation of CSCs, followed by their characterization in vitro, in vivo, and in patients' tissue samples. Fluorescent-activated cell sorting was used to isolate CD44+/CD271+ and CD44+/CD271- from two human HNSCC cell lines. Cell growth and self-renewal were measured with MTT and sphere/colony formation assays. Treatment-resistance was tested against chemotherapy (cisplatin and 5-fluorouracil) and ionizing radiation. Self-renewal, resistance, and stemness-related genes expression were measured with qRT-PCR. In vivo tumorigenicity was tested with an orthotopic immunodeficient mouse model of oral cancer. Finally, we examined the co-localization of CD44+/CD271+ in patients' tissue samples. We found that CD271+ cells were a subpopulation of CD44+ cells in human HNSCC cell lines and tissues. CD44+/CD271+ cells exhibited higher cell proliferation, sphere/colony formation, chemo- and radio-resistance, upregulation of CSCs-related genes, and in vivo tumorigenicity when compared to CD44+/CD271- or the parental cell line. These cell markers showed increased expression in patients with the increase of the tumor stage. In conclusion, using both CD44 and CD271 allowed the isolation of CSCs from HNSCC. These enriched CSCs will be more relevant in future treatment and HNSCC progression studies.

SEL120 - a First-in-Class CDK8/19 Inhibitor As a Novel Option for the Treatment of Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome - Data from Preclinical Studies and Introduction to a Phase Ib Clinical Trial

SEL120 - a First-in-Class CDK8/19 Inhibitor As a Novel Option for the Treatment of Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome - Data from Preclinical Studies and Introduction to a Phase Ib Clinical Trial

Reconstruction of Human AML Reveals Stem Cell Origin and Therapeutic Targets for Treatment Resistant CD34-/Lo MLL-Rearranged Leukemia

Identification of the origins of acute myeloid leukemia (AML) stem cells has been a holy grail for a better understanding of the developmental biology of the disease and the design of effective treatments. Studies using primary AML samples on patient-derived xenograft models identified AML stem cells in multiple different CD34/CD38 cellular fractions, which shared similar immunophenotypes of hematopoietic stem/progenitor cells including hematopoietic stem cells (HSCs), lymphoid-primed multipotent progenitors (LMPPs) and granulocyte-macrophage progenitors (GMPs). However inference of cells-of-origin based on the retrospective approach has major limitations as the reported HSC/LMPP/GMP-like AML stem cells are phenotypically different from their normal counterparts; and AML stem cells identified in late developmental stages do not necessarily maintain the same immunophenotypes of the disease initiating (pre-leukemic) cells, which can retain a relatively normal differentiation potential, and only their descendants acquire additional events becoming AML stem cells. While prospective disease modelling using mouse cells has provided unique insights into the potential origins of AML stem cells, human and mouse cells have different transformation requirements, distinct telomere biology, and a significant degree divergence of transcriptional regulation, chromatin state and gene regulatory networks that can profoundly affect their transformation potential and associated cancer biology. Therefore we reason that deconstruction of AML stem cell hierarchy from primary human samples followed by reconstruction of the corresponding human disease using candidate cell populations will give novel insights into this issue. Given that AML is a highly heterogeneous disease, our study initially focused on genetically well-defined MLL-rearranged AML frequently found in both infant and adult leukemia. By analyzing primary human AML patient samples, the current study unexpectedly revealed that AML stem cells driven by MLL fusions almost exclusively resided in immunophenotypically mature CD34-/loCD38+ compartments, as demonstrated by in vitro long-term culture initiating cells and in vivo limiting dilution xeno-transplantation assays into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using highly purified populations of hematopoietic stem/progenitor cells from normal human umbilical cord blood (UCB) to reconstruct of the human disease, we found that only human HSCs and common myeloid progenitors (CMPs), but not developmentally branched LMPPs and GMPs, could be disease initiating cells, transformed by MLL-fusions and induced leukemia in vivo, suggesting a key difference between human and mouse leukemia. As revealed by RNA-sequencing, LMPPs and GMPs failed to activate stem cell transcriptional programs and genes essential for MLL-leukemia including MEIS1 and HMGA2 in the early phase of transformation. MLL-fusions transformed HSCs and CMPs were immunophenotypically indistinguishable to leukemic cells of human MLL-AML patients, and also had an enrichment of AML stem cells in CD34-/loCD38+ compartment. Using machine learning, a specific gene signature could stratify patients into HSCs- and CMPs-derived AML, in which HSCs-derived AML showed a significantly poorer prognosis. To further investigate the biology of HSCs/CMPs-derived MLL-AML related to treatment responses, we subjected HSC/CMP MLL-AML leukemia with chemotherapeutic drugs and inhibitor of Bromodomain and Extra-Terminal motif (BET) currently used in AML trial. Interestingly, CMP-derived MLL-AML was treatment sensitive and PDX models transplanted with CMP-like AML samples could be largely cured by chemotherapy or BET inhibitor targeted therapy. In contrast, human HSCs-derived AML was highly resistant to the treatments. Strikingly, shRNA-mediated knockdown or pharmacological inhibition by fidaxomicin targeting ATP-binding cassette (ABC) transporters, ABCC3 that is highly expressed in HSCs-derived MLL-AML could re-sensitize the cells to the current chemotherapy. Together, the current study not only for the first time functionally identifies the origin of human MLL-AML stem cells, but also provides a new actionable venue for overcoming stem cells-associated treatment resistance by repositioning an anti-diarrhea drug, fidaxomicin currently available in the clinics. Disclosures Mufti: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding.