Selective advantage of mutant stem cells in human clonal hematopoiesis is associated with attenuated response to inflammation and aging.

Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging.

Transcriptomic classes of BCR-ABL1 lymphoblastic leukemia.

In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness and poor patient outcomes. A later arrest was associated with lineage fidelity, durable leukemia remissions and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ preleukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI efficacy are unexpected outcomes of the differentiation stage at which this leukemia transforms.

Human, mouse, and dog bone marrow show similar mesenchymal stromal cells within a distinctive microenvironment.

Bone marrow stromal cells (BMSCs) are a key part of the hematopoietic niche. Mouse and human BMSCs are recognized by different markers (LepR and NGFR/CD271, respectively). However, there has not been a detailed in situ comparison of both populations within the hematopoietic microenvironment. Moreover, dog BMSCs have not been characterized in situ by any of those markers. We conducted a systematic histopathological comparison of mouse, human, and dog BMSCs within their bone marrow architecture and microenvironment. Human and dog CD271+ BMSCs had a morphology, frequency, and distribution within trabecular bone marrow similar to those of mouse LepR+ BMSCs. However, mouse bone marrow had higher cellularity and megakaryocyte content. In conclusion, highly comparable bone marrow mesenchymal stromal cell distribution among the three species establishes the validity of using mouse and dog as a surrogate experimental model of hematopoietic stem cell-BMSC interactions. However, the distinct differences in adipocyte and megakaryocyte microenvironment content of mouse bone marrow and how they might influence hematopoietic stem cell interactions as compared with humans require further study.

Human Aging Alters the Spatial Organization between CD34+ Hematopoietic Cells and Adipocytes in Bone Marrow.

Age-related clonal hematopoiesis is a major risk factor for myeloid malignancy and myeloid skewing is a hallmark of aging. However, while it is known that non-cell-autonomous components of the microenvironment can also influence this risk, there have been few studies of how the spatial architecture of human bone marrow (BM) changes with aging. Here, we show that BM adiposity increases with age, which correlates with increased density of maturing myeloid cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) and an increased proportion of HSPCs adjacent to adipocytes. However, NGFR+ bone marrow stromal cell (NGFR+ BMSC) density and distance to HSPCs and vessels remained stable. Interestingly, we found that, upon aging, maturing myeloid cell density increases in hematopoietic areas surrounding adipocytes. We propose that increased adjacency to adipocytes in the BM microenvironment may influence myeloid skewing of aging HSPCs, contributing to age-related risk of myeloid malignancies.

Imaging methods used to study mouse and human HSC niches: Current and emerging technologies.

Bone marrow contains numerous different cell types arising from hematopoietic stem cells (HSCs) and non-hematopoietic mesenchymal/skeletal stem cells, in addition to other cell types such as endothelial cells- these non-hematopoietic cells are commonly referred to as stromal cells or microenvironment cells. HSC function is intimately linked to complex signals integrated by their niches, formed by combinations of hematopoietic and stromal cells. Studies of hematopoietic cells have been significantly advanced by flow cytometry methods, enabling the quantitation of each cell type in normal and perturbed situations, in addition to the isolation of these cells for molecular and functional studies. Less is known, however, about the specific niches for distinct developing hematopoietic lineages, or the changes occurring in the niche size and function in these distinct anatomical sites in the bone marrow under stress situations and ageing. Significant advances in imaging technology during the last decade have permitted studies of HSC niches in mice. Additional imaging technologies are emerging that will facilitate the study of human HSC niches in trephine BM biopsies. Here we provide an overview of imaging technologies used to study HSC niches, in addition to highlighting emerging technology that will help us to more precisely identify and characterize HSC niches in normal and diseased states.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

Retrospective cohort of pancreatic and Vater ampullary adenocarcinoma from a reference center in Mexico.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) and ampulla of Vater adenocarcinomas (AVAC) are periampullary tumors. These tumors have overlapping symptoms and a common treatment, but present differences in their survival and biology. No recent studies in Mexico have been published that describe the clinicopathological characteristics of these tumors. Therefore, the aim of this study was to describe the clinicopathological characteristics of PDAC and AVAC in patients at a reference center in Mexico. METHODS: A retrospective cohort of patients with PDAC or AVAC was analyzed at our institution (July 2007 to June 2016). Inferential analysis of the clinical data was performed with Student's t-test or a χ2 test with odds ratios (OR) and confidence intervals (CI), depending on the variables. Overall survival was compared using Kaplan-Meier curves with log-rank p values. RESULTS: Forty patients with PDAC and 76 with AVAC were analyzed, including 77 females and 39 males with a mean age of 60.6 years and a mean evolution time of 5.7 months. PDAC patients had more abdominal pain, a larger tumor size and more advanced stages than AVAC patients. In contrast, AVAC patients had more jaundice, a higher percentage of complete resections and higher overall survival. Up to 70% of patients were overweight. PDAC cohort included a higher proportion of smokers. CONCLUSIONS: Our cohort was slightly younger, had a larger percentage of females, and a greater percentage of obese patients than those in many international reports. A high proportion of PDAC patients are diagnosed in advanced stages and have a low likelihood of resectability.

From the bedside to the bench: new discoveries on blood cell fate and function.

Controversy and context: two words that exemplified this year's International Society for Experimental Hematology meeting. Leaders in the field of hematology from around the world gathered in San Diego in August of 2016 to discuss cutting-edge research on diverse topics such as hemoglobin switching, hematopoietic stem cell emergence, leukemogenesis, and aging. Major questions discussed included the "when, where, and how" of hematopoietic emergence, bone marrow residence, and disease origination. This meeting summary covers some of the conference highlights.

Human Mesenchymal Stem/Stromal Cells from Umbilical Cord Blood and Placenta Exhibit Similar Capacities to Promote Expansion of Hematopoietic Progenitor Cells In Vitro.

Mesenchymal stem/stromal cells (MSCs) from bone marrow (BM) have been used in coculture systems as a feeder layer for promoting the expansion of hematopoietic progenitor cells (HPCs) for hematopoietic cell transplantation. Because BM has some drawbacks, umbilical cord blood (UCB) and placenta (PL) have been proposed as possible alternative sources of MSCs. However, MSCs from UCB and PL sources have not been compared to determine which of these cell populations has the best capacity of promoting hematopoietic expansion. In this study, MSCs from UCB and PL were cultured under the same conditions to compare their capacities to support the expansion of HPCs in vitro. MSCs were cocultured with CD34+CD38-Lin- HPCs in the presence or absence of early acting cytokines. HPC expansion was analyzed through quantification of colony-forming cells (CFCs), long-term culture-initiating cells (LTC-ICs), and CD34+CD38-Lin- cells. MSCs from UCB and PL have similar capacities to increase HPC expansion, and this capacity is similar to that presented by BM-MSCs. Here, we are the first to determine that MSCs from UCB and PL have similar capacities to promote HPC expansion; however, PL is a better alternative source because MSCs can be obtained from a higher proportion of samples.

Bone Marrow Mesenchymal Stromal Cells from Clinical Scale Culture: In Vitro Evaluation of Their Differentiation, Hematopoietic Support, and Immunosuppressive Capacities.

The differentiation capacity, hematopoietic support, and immunomodulatory properties of human bone marrow mesenchymal stromal cells (BM-MSCs) make them attractive therapeutic agents for a wide range of diseases. Clinical scale cultures (CSCs) have been used to expand BM-MSCs for their use in cell therapy protocols; however, little is known about the functionality of the expanded cells. The main goal of the present study was to evaluate the functional characteristics of BM-MSCs expanded from CSCs to determine the quality of the cells for cellular therapy protocols. To address this issue, we analyzed the morphology, immunophenotype, differentiation potential (adipogenic, osteogenic and chondrogenic), hematopoietic support, and immunosuppressive capacity of BM-MSCs from short scale cultures (SSCs) and CSCs in a comparative manner. After 12 days of culture in CSCs (HYPERFlask System), BM-MSCs reached cell numbers of 125.52 × 10(6) ± 25.6 × 10(6) MSCs, which corresponded to the number of cells required for transplantation (∼1.7 × 10(6) MSCs/kg for a 70-kg patient). After expansion, BM-MSCs expressed the characteristic markers CD73, CD90, and CD105; however, expansion decreased their differentiation capacity toward the adipogenic, osteogenic, and chondrogenic lineages and their ability to inhibit T-cell proliferation compared with SSCs-MSCs. Importantly, CSCs-MSCs maintained the ability to support the proliferation and expansion of hematopoietic progenitor cells and the capacity to express the molecules, cytokines, and extracellular matrix proteins involved in the regulation of hematopoiesis. Our study highlights the need to evaluate the functional properties of the expanded BM-MSCs for verification of their quality for cell therapy protocols.

Beyond the Niche: Myelodysplastic Syndrome Topobiology in the Laboratory and in the Clinic.

We review the murine and human microenvironment and hematopoietic stem cell niche in the context of intact bone marrow architecture in man and mouse, both in normal and in myelodysplastic syndrome marrow. We propose that the complexity of the hematopoietic stem cell niche can usefully be approached in the context of its topobiology, and we provide a model that incorporates in vitro and in vivo models as well as in situ findings from intact human marrow to explain the changes seen in myelodysplastic syndrome patients. We highlight the clinical application of the study of the bone marrow microenvironment and its topobiology in myelodysplastic syndromes.

Hematopoiesis "awakens": Evolving technologies, the force behind them.

Amid the beauty of the Kyoto countryside, leaders in the field of hematology met at the 44th annual International Society for Experimental Hematology (ISEH) meeting in late September 2015. Led by ISEH President Paul Frenette and President-Elect David Traver, the meeting covered many aspects of hematopoiesis with a focus on technology. At the meeting, it became clear that the future of hematology is being shaped by innovations in single-cell "omics" and imaging approaches that will provide answers to age-old questions on cellular identity. In this meeting review, we highlight the advances presented in understanding the hematopoietic stem cell (HSC) niche, heterogeneity, stress response, epigenetics, and how these processes change from birth to old age.

Human mesenchymal stromal cells from adult and neonatal sources: a comparative in vitro analysis of their immunosuppressive properties against T cells.

Bone marrow-mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used in cell therapies as immune regulators for the treatment of graft-versus-host disease. We have previously characterized several biological properties of MSCs from placenta (PL) and umbilical cord blood (UCB), and compared them to those of BM-the gold standard. In the present study, we have compared MSCs from BM, UCB, and PL in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3(+) T cells. Our results confirm the immunosuppressive potential of BM-MSCs, and demonstrate that MSCs from UCB and, to a lesser extent PL, also have immunosuppressive potential. In contrast to PL-MSCs, BM-MSCs and UCB-MSCs significantly inhibited the proliferation of both CD4(+) and CD8(+) activated T cells in a cell-cell contact-dependent manner. Such a reduced proliferation in cell cocultures correlated with upregulation of programmed death ligand 1 on MSCs and cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) on T cells, and increased production of interferon-γ, interleukin-10, and prostaglandin E2. Importantly, and in contrast to PL-MSCs, both BM-MSCs and UCB-MSCs favored the generation of T-cell subsets displaying a regulatory phenotype CD4(+)CD25(+)CTLA-4(+). Our results indicate that, besides BM-MSCs, UCB-MSCs might be a potent and reliable candidate for future therapeutic applications.

El nicho de las células troncales: los secretos de su "código postal" / The niche of stem cells: the secrets of their "zip code"

El conocimiento de las células troncales ha salido de las cajas petri y los animales de experimentación para llegar a áreas que habían sido inexploradas por el conocimiento biológico. Líderes religiosos, presidentes y artistas han hablado y debatido sobre ellas. Las células troncales pueden ser consideradas como celebridades biológicas y, como cualquier celebridad, son escasas y difíciles de encontrar en cualquier otro sitio que no sean los titulares periodísticos. Las células troncales poseen varias características funcionales, dentro de las que destacan su capacidad de autorenovación y su gran potencial de proliferación y de diferenciación, características que las colocan en la mira tanto de la investigación básica como la investigación traslacional o aplicada. Las células troncales se localizan en áreas muy específicas dentro de los tejidos, denominadas como nichos. Los nichos proveen a las células troncales las condiciones necesarias para regular su fisiología preservar su estado de "célula troncal", además de que participan en la regulación de su proliferación y diferenciación. El conocer la localización de las células troncales y los mecanismos que las regulan en estos sitios, nos permitirá descubrir los secretos que guardan, para conocer su papel en la fisiopatología de las enfermedades y utilizarlos como posible blanco terapéutico, sacarlas de sus nichos más eficientemente para que sean accesibles para trasplantarlas e, incluso, producir células troncales en el laboratorio e inducir su diferenciación hacia tipos celulares específicos para su uso en protocolos de terapia celular y medicina regenerativa. En esta revisión nos enfocaremos a presentar el nicho de las células troncales hematopoyéticas (CTH) y la aplicación médica de este conocimiento.

Stem cells can be considered the new celebrities in biology and medicine, reaching areas that had been unexplored by biological knowledge. Religious leaders, presidents and artists have spoken and debated about them. Like any celebrity, they are rare and hard to find anywhere else other than news headlines. Self-renewal and their vast proliferation and differentiation potentials are among some characteristics that place them in the crosshairs of both basic and translational research. Stem cells are found in very specific areas within tissues, known as niches. Stem Cell niches provide the necessary conditions to regulate their physiology, preserving their "sternness" and controlling their proliferation and differentiation. Elucidating their "zip code" will lead us to know and manipulate their regulatory mechanisms. The stem cell niche is emerging as a new therapeutic target. We will discuss the hematopoietic stem cell niche and the clinical application of this knowledge.

In vitro evidence of the presence of mesenchymal stromal cells in cervical cancer and their role in protecting cancer cells from cytotoxic T cell activity.

Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSC populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papilloma virus, indicating that these cells are not infected by such a viral agent. Also, interestingly, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with the production of IL-10 in cell cocultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing downregulation of HLA class I molecules. This mechanism may have important implications in tumor growth.

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow.

Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal-Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.

In vitro effects of stromal cells expressing different levels of Jagged-1 and Delta-1 on the growth of primitive and intermediate CD34(+) cell subsets from human cord blood.

In trying to contribute to our knowledge on the role of Notch and its ligands within the human hematopoietic system, we have assessed the effects of the OP9 stroma cell line - naturally expressing Jagged-1 - transduced with either the Delta-1 gene (OP9-DL1 cells) or with vector alone (OP9-V), on the in vitro growth of two different hematopoietic cell populations. Primitive (CD34(+) CD38(-) Lin(-)) and intermediate (CD34(+) CD38(+) Lin(-)) CD34(+) cell subsets from human cord blood were cultured in the presence of 7 stimulatory cytokines under four different conditions: cytokines alone (control); cytokines and mesenchymal stromal cells; cytokines and OP9-V cells; cytokines and OP9-DL1 cells. Proliferation and expansion were determined after 7days of culture. Culture of CD34(+) CD38(-) Lin(-) cells in the presence of OP9-V or OP9-DL1 cells resulted in a significant increase in the production of new CD34(+) CD38(-) Lin(-) cells (expansion), which expressed increased levels of Notch-1; in contrast, production of total nucleated cells (proliferation) was reduced, as compared to control conditions. In cultures of CD34(+) CD38(+) Lin(-) cells established in the presence of OP9-V or OP9-DL1 cells, expansion was similar to that observed in control conditions, whereas proliferation was also reduced. Interestingly, in these latter cultures we observed production of CD34(+) CD38(-) Lin(-) cells. Our results indicate that, as compared to MSC, OP9 cells were more efficient at inducing self-renewal and/or de novo generation of primitive (CD34(+) CD38(-) Lin(-)) cells, and suggest that such effects were due, at least in part, to the presence of Jagged-1 and DL1.

Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood.

BACKGROUND AIMS: We have previously characterized the in vitro growth of two cord blood-derived hematopoietic cell populations in liquid cultures supplemented with recombinant cytokines. In the present study, we assessed the effects of bone marrow-derived mesenchymal stromal cells (MSC) on the growth of such cells. METHODS: CD34(+) CD38(+) Lin(-) and CD34(+) CD38(-) Lin(-) cells were obtained by negative selection, and cultured in the presence of marrow-derived MSC and/or early- and late-acting cytokines. Hematopoietic cell growth was assessed throughout a 30-day culture period. RESULTS: In the presence of MSC alone, both populations showed significant proliferation. Direct contact between MSC and CD34(+) cells was fundamental for optimal growth, especially for CD34(+) CD38(-) Lin(-) cells. In the presence of early-acting cytokines alone, cell growth was significantly higher than in cultures established with MSC but no cytokines. In cultures containing both MSC and early-acting cytokines, a further stimulation was observed only for CD34(+) CD38(-) Lin(-) cells. The cytokine cocktail containing both early- and late-acting cytokines was significantly more potent at inducing hematopoietic cell growth than the early-acting cytokine cocktail. When cultures were supplemented with early- and late-acting cytokines, MSC had no further effect on the growth of hematopoietic cells. CONCLUSIONS: MSC seem to play a key role, particularly on more primitive (CD34(+) CD38(-) Lin(-)) cells, only in the absence of cytokines or the presence of early-acting cytokines. When both early- and late-acting cytokines are present in culture, MSC seem to be unnecessary for optimal development of CFC and CD34(+) cells.

In vitro biology of human myeloid leukemia.

For about 40 years, the biology of human myeloid leukemia (ML) has been studied in different in vitro systems. Throughout this time, semisolid colony assays, Dexter-type long-term cultures and liquid suspension cultures have contributed to our understanding of the mechanisms involved in the origin and progression of this hematological disorder. By using such systems, it has been possible to identify the cells in which leukemia originates; to recognize a functional hierarchy within the hematopoietic system of leukemia patients; to identify factors, soluble and cell-associated, that regulate leukemic growth; and to study the effects of different antineoplastic drugs. Furthermore, in vitro systems for purging of leukemic cells have been developed. Still, many questions and problems remain unsolved regarding the biology of myeloid leukemia in vitro. This article presents a comprehensive review on the behavior of leukemic stem and progenitor cells, both from acute and chronic myeloid leukemia, in the different culture systems mentioned above.