Malformation of the endoplasmic reticulum system evolving into giant inclusions and Auer bodies in acute promyelocytic leukemia: an ultrastructural study of 6 cases.

The endoplasmic reticulum（ER）is the largest membranous network serving as a region for protein, lipid and steroid synthesis, transport and storage. Detailed information about ER-cisternae, ER-tubules and rough endoplasmic reticulum (rER) is scarce in human blood cells. This study describes a series of giant inclusions and Auer bodies in promyeloblasts in six patients with acute promyelocytic leukemia (APL), by light microscopy, transmission electron microscopy (TEM) and cytochemical stains. TEM revealed that giant inclusions and pro-Auer bodies were associated with rER and surrounded by tubular structures composed of degenerated or redundant membrane in promyeloblasts, which corresponded with elements of the ER system. This paper reveals that in the promyeloblasts of APL, ER is the source of and transforms progressively into giant inclusions and Auer bodies.

Structural characterization and origin of surface vesicles in monocytes: another membranous pathway from cytoplasm to cell surface.

The monocytes in acute monocytic leukemia (AML-M5b) were analyzed by scanning and transmission electron microscopy (SEM and TEM) to understand more fully their structure and origin. By SEM, monocytes exhibited localized expansions of the surface, some of which appeared to bud off as surface vesicles (SVs). Filopodial processes and pseudopodia were also present. TEM demonstrated that the SVs were composed of a double-membrane at the pole away from the cell body, and a single membrane nearer to the cell body. In the peripheral cytoplasm, intracellular vesicles (IVs) had the appearance of vacuoles and were enclosed by single membranes. Most SVs were characterized by a notch as a rER edge and an expanded head. Filopodial processes had the same thickness of 40 nm as the SV walls, which suggested a close developmental relationship between the two. Pseudopodia between SVs were irregular in size. Rod-like rER cisternae were prominent in the peripheral cytoplasm and some showed a close physical juxtaposition as to suggest a transition from rER to IVs to SVs. Ultrastructural cytochemistry demonstrated activity of 5'-nucleotidase over rER, SVs, filopodial processes and pseudopodia, and a patchy reaction over other areas of plasma membrane. Overall, the results indicated that rER transforms into SVs, filopodial processes and pseudopodia, as a way of integrating cytoplasmic membranes into the plasma membrane.

Ultrastructural analysis of nucleated erythrocyte in patients with autoimmune hemolytic anemia (AIHA).

Autoimmune hemolytic anemia (AIHA) is a group of diseases characterized by immune-mediated lysis of mature red blood cells (RBCs). It is mainly classified into primary and secondary types based on etiology and mechanisms underlying autoantibody production. AIHA is diagnosed using morphological observation of bone marrow smears under a light microscope and monospecific direct antiglobulin test to detect hemolysis. Here, we retrospectively studied ultrastructural abnormalities of nucleated erythroid cells in bone marrows from 10 patients with AIHA using transmission electron microscopy. Our results revealed severe damage and injury to nucleated erythroid cells, including morphological irregularity, pyknosis, karyolysis, expansion of perinuclear cisternae and cytoplasmic lysis. These results indicate that aberrant immunity attacks not only mature RBCs but also nucleated erythroid cells, and ineffective hematopoiesis is partly involved in the pathogenesis of AIHA.

Development of Auer bodies from giant inclusions associated with rough endoplasmic reticulum in acute promyelocytic leukemia.

Giant inclusions and Auer bodies in promyeloblasts were investigated in a study which included transmission electron microscopy (TEM) for morphology and ultrastructural cytochemistry for myeloperoxidase in 10 patients with acute promyelocytic leukemia (APL). Ultrastructural cytochemistry demonstrated positive myeloperoxidase reactivity in giant inclusions, expanded rER cisternae, Auer bodies and primary granules. TEM revealed that giant inclusions were adorned by degenerated rER membrane, some of them sharing features with Auer bodies. We hypothesize a novel origin for Auer body development in promyeloblasts of APL, namely that they originate from peroxidase-positive and expanded rER cisternae, and that primary granules were directly released from these expanded rER elements, bypassing the Golgi apparatus.

Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes syndrome: a case report.

A biopsy of gastrocnemius muscle from a patient with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome was studied histologically in semithin sections stained by hematoxylin-and-eosin (H&E) and toluidine blue, and ultrathin sections by transmission electron microscopy (TEM). H&E stain demonstrated typical ragged-red fibers (RRFs) and affected fibers in fascicles. Toluidine-blue stain showed an irregular meshwork in the center of RRFs. TEM demonstrated damaged myofibrils and variations in mitochondrial structure in RRFs and affected fibers. Dense mitochondria were compacted with cristae and pleomorphic electron-dense inclusions. Lucent mitochondria included paracrystalline inclusions with a parking lot appearance. At high magnification, the paracrystalline inclusions were composed of plates that paralleled and connected with mitochondrial cristae. These observations indicated that electron-dense granular and paracrystalline inclusions resulted from cristal degeneration and overlapping in mitochondria in MELAS syndrome.

Ultrastructural characteristics of erythroid cells in congenital dyserythropoietic anemia type II, with a focus on peripheral cisternae and double membranes.

Peripheral cisternae and double membranes (PCDMs) in erythroid cells are a landmark of type II congenital dyserythropoietic anemia (CDA). To gain further insights into the mechanism of dyserythropoiesis, erythroblasts and erythrocytes in bone marrow were studied in 22 Chinese patients with CDA Ⅱ by transmission electron microscopy. The study demonstrated an increase in all patients in erythroblasts with PCDMs with development from pro-erythroblast to red blood cells. PCDMs often connected with cisternae of endoplasmic reticulum (ER) and the perinuclear space, and were accompanied by karyopyknosis, karyolysis and disruption in polychromatic and orthochromatic erythroblasts. The results suggest that PCDMs are transformed from ER during erythropoiesis and participate in the dissolution and deletion of late erythroid cells in patients with CDA II.

Deciphering the Heterogeneity of Mitochondrial Functions During Hematopoietic Lineage Differentiation.

BACKGROUND: The heterogeneity of mitochondrial function is an important feature of hematopoietic cell lineage differentiation, but its stage wise contribution is not adequately studied. To establish a model to compare the lineage differentiation of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, the mitochondrial mass (MM), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitophagy level were analyzed. RESULTS AND DISCUSSION: HSCs had lower mitochondrial metabolic activity than committed progenitor populations, indicated by lower MM, MMP, and ROS and higher mitophagy. HPC1s shared more stem cell characteristics than HPC2s and committed progenitor populations in terms of mitochondrial number and function. The mitochondrial metabolism of mature blood cells had greater heterogeneity than hematopoietic stem and progenitor cells, with granulocytes being similar to monocytes. Moreover, HSCs exhibited heterogeneity in the selection of mitophagy-related PINK1/PARK2, BNIP3/NIX, and FUNDC1 pathways. Myeloid differentiation had greater morphological and functional heterogeneity of hematopoietic cells than lymphoid differentiation. Additionally, leukemia stem cells had higher aerobic metabolism and better stem cell function through elevated mitophagy than normal hematopoietic cells. ROS and MMP levels in differentiated leukemia cells were higher, but the level of mitophagy was lower than in differentiated hematopoietic cells. CONCLUSION: This study provides a complete set of methods and basic reference values for the systematic study of the mitochondrial metabolic function of different types of hematopoietic cells under physiological and pathological conditions. The findings contribute to the future research of tumor and aging based on mitochondrial metabolism.

Rapid and non-invasive discrimination of acute leukemia bone marrow supernatants by Raman spectroscopy and multivariate statistical analysis.

A simple and non-invasive detection method for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) was established by systematically investigating the characteristics of bone marrow supernatants from 61 AML patients, 22 ALL patients, and 5 volunteers without hematological tumors by Raman spectroscopy and orthogonal partial least squares discriminant analysis (OPLS-DA). The control group could be well distinguished from the AML and ALL groups by Raman peaks of 859, 1031, 1437, 1443, 1446, 1579, and 1603 cm-1 and from the AML subtypes groups (AML-M2, AML-M3, AML-M4, and AML-M5) by the Raman peaks of 859, 1221, 1230, 1437, 1443, and 1603 cm-1, indicating high sensitivity and specificity of the method. Potentially important variables of acute leukemia (AL) prognosis, such as cholesterol, high-density lipoprotein, low-density lipoprotein, adenosine deaminase, and hemoglobin, could be effectively identified by Raman peaks of 1437, 1443, and 1579 cm-1. Therefore, Raman spectroscopy can be considered as a new non-invasive clinical tool for the detection of different types of AL and can be used to correlate biochemical parameters of AL patients with the classification and prognosis of AL.

Bone marrow mesenchymal cells: polymorphism associated with transformation of rough endoplasmic reticulum.

To understand the behavior and function of bone-marrow mesenchymal cells (BMMCs), we overviewed the morphological presentation of BMMCs in bone-marrow granules (b-BMMCs), isolated BMMCs (i-BMMCs), and BMMCs (c-BMMCs) cultured in H4434 methylcellulose semisolid and MEM media. All samples were derived from bone-marrow aspirates of 30 patients with hematocytopenia. Light microscopy exhibited b-BMMCs and i-BMMCs characterized by abundant cytoplasm and irregular shape in bone-marrow smears, as well as c-BMMCs in culture conditions. Scanning electron microscopy demonstrated cultured c-BMMCs with a sheet-like feature enveloping hematopoietic cells. Transmission electron microscopy revealed b-BMMCs constructing a honeycomb-like structure by thin bifurcate processes among hematopoietic cells. Furthermore, i-BMMCs had bifurcate parapodiums on the surface and prominent rough endoplasmic reticulum (rER) connected with the plasmalemma of the parapodiums. The detailed images suggested that rER may serve as a membrane resource for plasmalemmal expansion in BMMCs in bone marrow.

Ultrastructural alterations of megakaryocytes in thrombocytopenia: A review of 43 cases.

Thrombocytopenia is a frequent occurrence in a variety of hematopoietic diseases; however, the details of the mechanism leading to low platelet count remain elusive. Megakaryocytes are a series of progenitor cells responsible for the production of platelets. Alterations in megakaryocytes in the bone marrow are a causative factor resulting in thrombocytopenia in varied diseases. Based on ultrastructural analysis of incidentally encountered megakaryocytes in 43 patients with blood diseases marked by low platelet counts, electron micrographs demonstrated that aberrant megakaryocytes predominated in idiopathic thrombocytopenic purpura, aplastic anemia, and myelodysplastic syndrome; autophagy, apoptosis, and cellular damage in megakaryocytes were a prominent feature in aplastic anemia. On the other hand, poorly differentiated megakaryocytes predominated in acute megakaryoblastic leukemia (AMKL) although damaged megakaryocytes were seen in non-AMKL acute leukemia. This paper documents the ultrastructural alterations of megakaryocytes associated with thrombocytopenia and reveals distinctive features for particular blood diseases. A comment is made on future avenues of research emphasizing membrane fusion proteins.

Comparative analysis of full-length transcriptomes based on hybrid population reveals regulatory mechanisms of anthocyanin biosynthesis in sweet potato (Ipomoea batatas (L.) Lam).

BACKGROUND: Sweet potato (Ipomoea batatas (L.) Lam.) is a highly heterozygous autohexaploid crop with high yield and high anthocyanin content. Purple sweet potato is the main source of anthocyanins, and the mechanism of anthocyanin biosynthesis in storage roots has not been fully revealed. RESULTS: In order to reveal the mechanism of anthocyanin biosynthesis and identify new homologous genes involved in anthocyanin biosynthesis in the storage roots of sweet potato, we used Ningzishu 1 and Jizishu 2 as parents to construct a F1 hybrid population. Seven anthocyanin-containing lines and three anthocyanin-free lines were selected for full-length and second-generation transcriptome analyses. A total of 598,375 circular consensus sequencing reads were identified from full-length transcriptome sequencing. After analysis and correction of second-generation transcriptome data, 41,356 transcripts and 18,176 unigenes were obtained. Through a comparative analysis between anthocyanin-containing and anthocyanin-free groups 2329 unigenes were found to be significantly differentially expressed, of which 1235 were significantly up-regulated and 1094 were significantly down-regulated. GO enrichment analysis showed that the differentially expressed unigenes were significantly enriched in molecular function and biological process. KEGG enrichment analysis showed that the up-regulated unigenes were significantly enriched in the flavonoid biosynthesis and phenylpropanoid biosynthesis pathways, and the down-regulated unigenes were significantly enriched in the plant hormone signal transduction pathway. Weighted gene co-expression network analysis of differentially expressed unigenes revealed that anthocyanin biosynthesis genes were co-expressed with transcription factors such as MYB, bHLH and WRKY at the transcription level. CONCLUSIONS: Our study will shed light on the regulatory mechanism of anthocyanin biosynthesis in sweet potato storage roots at the transcriptome level.

Foam cell origination from degenerated vascular smooth muscle cells in atherosclerosis: An ultrastructural study on hyperlipidemic rabbits.

To clarify foam cell origination in atherosclerosis, a series of morphologic and ultrastructural alterations of vascular smooth muscle cells (VSMCs) and foam cells were studied by light and electron microscopy in atherosclerotic aortas from hyperlipidemic rabbits induced for 5 weeks. The study exhibited that VSMCs were severely degenerated and damaged, including irregular shapes, expanded mitochondria, aplenty lipid droplets, and disarranged myofilaments in cytoplasm in media adjacent to atheromatic bottoms. Most lipid laden cells shared interphase structures of VSMCs and foam cells, and some dissolved spindle cells contained lipid droplets, lipofuscin, and rod-like CCs in cytoplasm also. The result demonstrated that VSMCs were degenerated and transformed into foam cells in atherosclerosis, which was responsible for the accumulation of lipid and cholesterol crystals in atherosclerotic arteries.

One cell one niche: hematopoietic microenvironments constructed by bone marrow stromal cells with fibroblastic and histiocytic features.

Hematopoietic microenvironments have been extensively studied, especially focusing on regulation of hematopoietic stem cells (HSCs) in HSC niche following progress of molecular biology in resent years. Based on prior morphological achievements from 1970s, the characteristics of cellular compartments and bone marrow stromal cells (BMSCs) were studied ultrastructurally in human and mice bone marrow in the present study. The samples, human bone marrow granules, were collected from bone marrow aspirations (BMAs) of 20 patients with hematocytopenia and isolated BMSCs were found undesignedly in nucleated cells of BMAs of the patients. Femoral bone marrow samples were collected from 6-week-old three sacrificed mice. Detailed images illustrated maturing hematopoietic cells harbored individually in honeycomb-like microenvironment constituted by BMSCs that shared of fibroblastic and histiocytic characteristics in hematopoietic microenvironments of human and mice bone marrow.

Thrombopoietin knock-in augments platelet generation from human embryonic stem cells.

BACKGROUND: Refinement of therapeutic-scale platelet production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy. However, procedures for cost-effective and scalable platelet generation remain to be established. METHODS: In this study, we established human embryonic stem cell (hESC) lines containing knock-in of thrombopoietin (TPO) via CRISPR/Cas9-mediated genome editing. The expression and secretion of TPO was detected by western blotting and enzyme-linked immunosorbent assay. Then, we tested the potency for hematopoietic differentiation by coculturing the cells with mAGM-S3 cells and measured the generation of CD43+ and CD45+ hematopoietic progenitor cells (HPCs). The potency for megakaryocytic differentiation and platelet generation of TPO knock-in hESCs were further detected by measuring the expression of CD41a and CD42b. The morphology and function of platelets were analyzed with electronic microscopy and aggregation assay. RESULTS: The TPO gene was successfully inserted into the AAVS1 locus of the hESC genome and two cell lines with stable TPO expression and secretion were established. TPO knock-in exerts minimal effects on pluripotency but enhances early hematopoiesis and generation of more HPCs. More importantly, upon its knock-in, TPO augments megakaryocytic differentiation and platelet generation. In addition, the platelets derived from hESCs in vitro are functionally and morphologically comparable to those found in peripheral blood. Furthermore, TPO knock-in can partially replace the large quantities of extrinsic TPO necessary for megakaryocytic differentiation and platelet generation. CONCLUSIONS: Our results demonstrate that autonomous production of cytokines in hESCs may become a powerful approach for cost-effective and large-scale platelet generation in translational medicine.

PDK1 regulates definitive HSCs via the FOXO pathway during murine fetal liver hematopoiesis.

PDK1 (phosphoinositide dependent kinase-1) plays an important regulatory role in B cells, T cells and platelets. Less is known about how PDK1 acts in hematopoietic stem cells (HSCs), especially in the fetal liver (FL) during embryonic hematopoiesis, as the FL is the primary fetal hematopoietic organ and the main site of HSC expansion and differentiation. Here, we deleted the PDK1 gene in hematopoietic cells by crossing Vav-Cre transgenic mice with PDK1f/f mice. Using a transplantation assay, we found that HSCs from the E15.5 FL of Vav-Cre;PDK1f/f embryos are severely impaired compared when compared with HSCs from PDK1f/f or PDK1f/+ FLs. Additionally, we found that there were more FL HSCs in an apoptotic state and active cell cycle in PDK1-deficient embryos than in control embryos. By comparing the expression profiles of FL-derived LSKs in Vav-Cre;PDK1f/f embryos to the controls, we found that the BH3-only protein PUMA and the cyclin family proteins were expressed higher in the Vav-Cre;PDK1f/f group, which may account for the increased apoptosis and activated cell cycle in the deficient HSCs. Furthermore, we demonstrated that the expression of FoxO3a was higher in PDK1-deficient LSKs, indicating that the Akt-FoxO3a-PUMA axis may participate in regulating LSKs apoptosis in the E15.5 FL. In contrast, FoxO1 expression was lower in PDK1-deficient LSK cells, suggesting that Akt-FoxO1-CCND may regulate the HSC cell cycle. Taken together, our findings support a critical role for PDK1 in maintaining FL hematopoiesis via regulating apoptosis and cell cycle of definitive hematopoiesis by the Akt-FOXO signaling pathways.

A novel anemia associated with membranous cytoplasm degeneration in 16 patients: an ultrastructural study.

Sixteen patients with mild anemia and hemolysis were difficult to be classified into any known category based on laboratory examinations and light microscopy. To make a definite diagnosis and investigate the pathomechanism, ultrastructural study was performed on erythroid cells from 16 patients. Transmission electron microscopy demonstrated a series of alterations of cytoplasm, including cytoplasm sequestration, membranous transformation, and degeneration in erythroblasts and reticulocytes at different stages. The affected erythroblasts were usually complicated with chromatin condensation, karyorrhexis, nuclear membrane lysis, and megaloblastic changes. The reticulocytes with the cytoplasm alterations had a huge size from 10 um to 15 um in diameter. The membranous cytoplasm degeneration revealed a unique pathomechanism of dyserythropoiesis and ineffective erythropoiesis in 16 patients with anemia, and suggested a novel anemia category though more details remained to be investigated.

[Ultrastructure and Raman Spectral Characteristics of Two Kinds of Acute Myeloid Leukemia Cells].

OBJECTIVE: To investigate the Raman spectral characteristics of leukemia cells from 4 patients with acute promyelocytic leukemia (M3) and 3 patients with acute monoblastic leukemia (M5), establish a novel Raman label-free method to distinguish 2 kinds of acute myeloid leukemia cells so as to provide basis for clinical research. METHODS: Leukemia cells were collected from bone marrow of above-mentioned patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 30-50 cells from each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA) and cluster analysis, and the spectra of leukemia cells from 7 patients were analyzed and classified. Characteristics of Raman spectra were analyzed combining with ultrastructure of leukemia cells. RESULTS: There were significant differences between Raman spectra of 2 kinds of leukemia cells. Compared with acute monoblastic leukemia cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks in 622, 643, 757, 852, 1003, 1033, 1117, 1157, 1173, 1208, 1340, 1551, 1581 cm-1. The diagnostic models established by PCA-DFA and cluster analysis could successfully classify these Raman spectra of different samples with a high accuracy of 100% (233/233). The model was evaluated by "Leave-one-out" cross-validation and reached a high accuracy of 97% (226/233). CONCLUSION: The level of macromolecules of M3 cells is higher than that of M5. The diagnostic models established by PCA-DFA can classify these Raman spectra of different cells with a high accuracy. Raman spectra shows consistent result with ultrastructure by TEM.