Full-length, single-cell RNA-sequencing of human bone marrow subpopulations reveals hidden complexity

Bone marrow progenitor cell differentiation has frequently been used as a model for studying cellular plasticity and cell-fate decisions. Recent analysis at the level of single-cells has expanded knowledge of the transcriptional landscape of human hematopoietic cell lineages. Using single-molecule real-time (SMRT) full-length RNA sequencing, we have previously shown that human bone marrow lineage-negative (Lin-neg) cell populations contain a surprisingly diverse set of mRNA isoforms. Here, we report from single cell, full-length RNA sequencing that this diversity is also reflected at the single-cell level. From fresh human bone marrow unselected and lineage-negative progenitor cells were isolated by droplet-based single-cell selection (10xGenomics). The single cell-derived mRNAs were analyzed by full-length SMRT and short-read sequencing. In both samples we detected an average of 8000 different genes using short-read sequencing. Differential expression analysis arranged the single-cells of the total bone marrow into only four clusters whereas the Lin-neg population was much more diverse with nine clusters. mRNA isoform analysis of the single-cell populations using full-length sequencing revealed that Lin-neg cells contain on average 24% more novel splice variants than the total bone marrow cells. Interestingly, among the most frequent genes expressing novel isoforms were members of the spliceosome, e.g. HNRNPs, DEAD box helicases and SRSFs. Mapping the isoforms from all genes to the cell type clusters revealed that total bone marrow cells express novel isoforms only in a small subset of clusters. On the other hand, lineage-negative progenitor cells expressing novel isoforms were present in nearly all subpopulations. In conclusion, on a single-cell level lineage-negative cells express a higher diversity of genes and more alternatively spliced novel isoforms suggesting that cells in this subpopulation are poised for different fates.

Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

Trilaciclib dose selection: an integrated pharmacokinetic and pharmacodynamic analysis of preclinical data and Phase Ib/IIa studies in patients with extensive-stage small cell lung cancer

Purpose Trilaciclib is a first-in-class CDK4/6 inhibitor that transiently arrests hematopoietic stem and progenitor cells (HSPCs) in the G1 phase of the cell cycle to preserve them from chemotherapy-induced damage (myelopreservation). We report integrated analyses of preclinical and clinical data that informed selection of the recommended Phase II dose (RP2D) used in trilaciclib trials in extensive-stage small cell lung cancer (ES-SCLC). Methods A semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model developed from preclinical data guided selection of an optimal dose for G1 bone marrow arrest in a first-in-human Phase I study (G1T28-1-01). PK, PD, safety, and efficacy data from G1T28-1-01 and two Phase Ib/IIa studies (G1T28-02/-03) in ES-SCLC were analyzed to support RP2D selection. Results Model simulation of bone marrow arrest based on preclinical data predicted that a ≥ 192 mg/m2 dose would induce a 40–50% decrease in total bone marrow proliferation in humans and almost 100% cell cycle arrest of cycling HSPCs. Consistent with this model, analysis of bone marrow aspirates in healthy volunteers after trilaciclib 192 mg/m2 administration demonstrated almost 100% G1 arrest in HSPCs and 40% decrease in total bone marrow proliferation, with minimal toxicity. G1T28-02/-03 reported similar PK parameters with trilaciclib 200 mg/m2 but slightly lower exposures than expected compared with healthy volunteers; consequently, 240 and 280 mg/m2 doses were also tested to match healthy volunteer exposures. Based on PK and relevant safety data, 240 mg/m2 was selected as the RP2D, which was also favored by myelopreservation endpoints in G1T28-02/-03. Conclusion Integrated PK/PD, safety, and efficacy data support 240 mg/m2 as the RP2D for trilaciclib. ClinicalTrials.gov Identifiers NCT02243150; NCT02499770; NCT02514447.

The state of the red bone marrow in rats depending on age in case of poisoning with lead acetate and potassium dichromate

Introduction: The current ecological situation is described by a rapid increase in air pollution through various chemical emissions. Aim: The study aims to define how the level of lead and chromium compounds affects the parameters of red bone marrow and blood in animals and how these processes depend on their age. Material and methods: To study the toxic intraday effect of heavy metals, a metal probe was used for combined priming of rats with lead acetate and potassium dichromate. The indicators were analyzed with the use of a cytogram, and the indices of the ratio between young and mature forms were calculated. The obtained factual material was subjected to computer processing with the calculation of the Student’s criterion and confidence intervals. Results and discussion: It was established that in young rats there is an accumulation of polychromatophilic cells with a large amount of hemoglobin, which leads to a violation of their maturation – with that, the subsequent stage of division passes very quickly, when, upon transforming into orthochromatic normoblasts, they pass into the blood after 15–20 h, forming a variant of terminal division. In old animals, there was a decrease in total bone marrow cellularity and severe anemia. Conclusions: After the experiments and profound analysis, the author of the study defined the difference of toxic effects on young and old animals.

How Relevant Are Bone Marrow-Derived Mast Cells (BMMCs) as Models for Tissue Mast Cells? A Comparative Transcriptome Analysis of BMMCs and Peritoneal Mast Cells

Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs.

Low-Dose Epo after Tgfβ Blockade Triggers Robust Erythropoiesis and Increased RBC Production

Background: Therapy for chronic anemias is limited to RBC transfusions and Erythropoiesis Stimulating Agents (ESA) which often work on only transiently or not at all. New approaches to treat chronic anemia are needed but development has been limited by our incomplete understanding of erythropoiesis, most of which relates to the terminal maturation of erythroid precursors. Erythropoietin (Epo) acts during a very narrow window of erythropoiesis, well after progenitor commitment to an exclusively erythroid fate. It is not known if the final steps of RBC maturation are coupled to the earlier stages of hematopoietic stem and progenitor cell (HSPC) differentiation; a process that begins almost three weeks earlier when an HSC starts its march towards committed RBC precursors via a series of branching cell fate decisions.We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Results: We deleted TGFβ1 in megakaryocytes (TGFβ1ΔMk/ΔMk) and found that peripheral blood counts were normal in TGFβ1ΔMk/ΔMkmice compared to TGFβ1FL/FLcontrols despite the pool of primitive hematopoietic cells being expanded (Fig. 1a). Similarly total bone marrow cellularity was normal in TGFβ1ΔMk/ΔMkmice (Fig. 1b). Excess HSCs in TGFβ1ΔMk/ΔMkmice appeared capable of robust differentiation because the number of immature lineage-negative (Linneg) hematopoietic progenitor cells was increased in the marrows of TGFβ1ΔMk/ΔMkmice (Fig. 1c). Thus, it remained unexplained why the expanded number of HSPCs (Fig. 1d) do not increase blood counts and marrow cellularity. We hypothesized that the excess progenitors observed in the TGFβ1ΔMk/ΔMkmice failed to increase blood counts because their progeny were unneeded, and inadequately supported by homeostatic levels of late-acting cytokines. Indeed, bone marrow apoptosis was increased in the TGFβ1ΔMk/ΔMkmice compared to controls, as reported by AnnexinV (AV) binding (Fig. 1e-f). Apoptosis of lineage-marker negative (Linneg), Kit+Sca1neg(LKSneg) HPCs and LKS+HSPCs was rare in both TGFβ1ΔMk/ΔMkmice and littermate controls (Fig. 1g). These results suggest that excess, hematopoietic precursors present in the TGFβ1ΔMk/ΔMkmice are pruned by apoptosis during hematopoietic differentiation. We found 10-fold apoptosis in TGFβ1ΔMk/ΔMkprecursors populations BM (Fig. 1h). Epo levels were normal in the serum of these mice, we reasoned that the excess, unneeded cells were not supported physiologic Epo levels. To test this, we treated mice with exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (300U/kg)(Fig. 1i-j). Whereas TGFβ1Flox/Floxmice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMkmice responded with reticulocytosis and erythrocytosis within 6 days (Fig. 1k-l). In contrast, treatment of mice with TGFβ1 worsened the erythroid apoptosis observed in TGFβ1ΔMk/ΔMkmice and caused mild anemia. These results suggest that erythropoiesis is subject to modular regulation with megakaryocytic TGFβ1 constraining the pool of erythroid committed progenitors that are then licensed to mature via Epo signaling. We thought that blockade of TGFβ signaling could phenocopy these effects by inducing overproduction of erythroid committed precursors. To test this, we pre-treated B6 mice with a TGFβ1 neutralizing antibody (1D11) or non-targeting, isotype control antibody (13C4) and then either PBS or low-dose Epo (Fig. 1m). TGFβ neutralization by 1D11 reduced pSmad2/3 MFI in HSPCs in wild-type mice whereas the 13C4 control had no effect, demonstrating on-target activity (Fig. 1n). Low-dose Epo triggered a brisk erythropoietic response in mice treated with 1D11 but not those treated with the 13C4 control (Fig. 1o). Exogenous Epo rescued the erythroid precursor dropout observed in B6 mice treated with 1D11 but did not affect the low apoptosis observed in mice treated with the 13C4 control (Fig. 1p-r). Therefore, the boundary of megakaryocytic TGFβ1 activity is compartmentalized within the marrow with predominant effects on immature HSPCs while excluding their progeny (Fig. 4s). Conclusion: This work also promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Waldenström Macroglobulinemia: Unusual Presentation With Cast Nephropathy/Light Chain Tubulopathy

Cast Nephropathy/Light chain tubulopathy is usually present in patients with multiple myeloma and is very rare in patients with Waldenstrom Macroglobulinemia. There are very few case reports mentioned in the literature. We present an interesting case of Cast Nephropathy and light chain tubulopathy in an 81-year-old female patient with Waldenstrom Macroglobulinemia who required medical attention for worsening renal failure. Serum protein electrophoresis/Immunofixation showed IgM Kappa monoclonal gammopathy. Renal biopsy was remarkable for cast nephropathy and light chain tubulopathy. Furthermore on bone marrow biopsy a low grade B cell lymphoproliferative disorder with plasmacytic differentiation was present. This was most consistent with lymphoplasmacytic lymphoma, accounting for 50-60 percent of total bone marrow cellularity, in a hypercellular (60-80 percent) bone marrow.