Differentiation Of Multipotent Progenitors Research Articles

Acute malaria suppresses the B lymphocytic niche in the bone marrow through the alteration of CXCL12-abundant reticular cells.

Bone marrow is a dynamic organ composed of stem cells that constantly receive signals from stromal cells and other hematopoietic cells in the niches of the bone marrow to maintain hematopoiesis and generate immune cells. Perturbation of the bone marrow microenvironment by infection and inflammation affects hematopoiesis and may affect immune cell development. Little is known about the effect of malaria on the bone marrow stromal cells that govern the hematopoietic stem cell (HSC) niche. In this study, we demonstrate that the mesenchymal stromal CXCL12-abundant reticular (CAR) cell population is reduced during acute malaria infection. The reduction of CXCL12 and IL-7 signals in the bone marrow impairs the lymphopoietic niche, leading to the depletion of common lymphoid progenitors, B cell progenitors and mature B cells, including plasma cells in the bone marrow. We found that IFNγ is responsible for the upregulation of Sca1 on CAR cells, yet the decline in CAR cell and B cell populations in the bone marrow is IFNγ-independent. In contrast to the decline in B cell populations, HSCs and multipotent progenitors increased with expansion of myelopoiesis and erythropoiesis, indicating a bias in the differentiation of multipotent progenitors during malaria infection. These findings suggest that malaria may affect host immunity by modulating the bone marrow niche.

Inhibition of TGF\u03b2 improves hematopoietic stem cell niche and ameliorates cancer-related anemia

BackgroundCancer cachexia is a wasting syndrome that is quite common in terminal-stage cancer patients. Cancer-related anemia is one of the main features of cancer cachexia and mostly results in a poor prognosis. The disadvantages of the current therapies are obvious, but few new treatments have been developed because the pathological mechanism remains unclear.MethodsC57BL/6 mice were subcutaneously injected with Lewis lung carcinoma cells to generate a cancer-related anemia model. The treated group received daily intraperitoneal injections of SB505124. Blood parameters were determined with a routine blood counting analyzer. Erythroid cells and hematopoietic stem/progenitor cells were analyzed by flow cytometry. The microarchitecture changes of the femurs were determined by micro-computed tomography scans. Smad2/3 phosphorylation was analyzed by immunofluorescence and Western blotting. The changes in the hematopoietic stem cell niche were revealed by qPCR analysis of both fibrosis-related genes and hematopoietic genes, fibroblastic colony-forming unit assays, and lineage differentiation of mesenchymal stromal cells.ResultsThe mouse model exhibited hematopoietic suppression, marked by a decrease of erythrocytes in the peripheral blood, as well as an increase of immature erythroblasts and reduced differentiation of multipotent progenitors in the bone marrow. The ratio of bone volume/total volume, trabecular number, and cortical wall thickness all appeared to decrease, and the increased osteoclast number has led to the release of latent TGFβ and TGFβ signaling over-activation. Excessive TGFβ deteriorated the hematopoietic stem cell niche, inducing fibrosis of the bone marrow as well as the transition of mesenchymal stromal cells. Treatment with SB505124, a small-molecule inhibitor of TGFβ signaling, significantly attenuated the symptoms of cancer-related anemia in this model, as evidenced by the increase of erythrocytes in the peripheral blood and the normalized proportion of erythroblast cell clusters. Meanwhile, hindered hematopoiesis and deteriorated hematopoietic stem cell niche were also shown to be restored with SB505124 treatment.ConclusionThis study investigated the role of TGFβ released by bone remodeling in the progression of cancer-related anemia and revealed a potential therapeutic approach for relieving defects in hematopoiesis.

Crispr/Cas9-Induced DNMT3A Mutations in the K562 Human Leukemic Cell Line As a Model of DNMT3A-Mutated Leukemogenesis

Background:DNA methyltransferase 3A(DNMT3A) is a member of the DNA methyltransferase family primarily involved in de novo gene methylation. Mutations in DNMT3A are associated with a wide range of hematological malignancies, most frequently acute myeloid leukemia (AML). DNMT3A mutations are thought to produce a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. Mutations in DNMT3A often coexist with secondary lesions in leukemia-related genes such as NPM1 and FLT3 (Ley T et al., N Engl J Med, 2010). Furthermore, healthy individuals harboring DNMT3A-driven clonal hematopoiesis are at increased risk of future hematologic malignancies and all-cause mortality (Jaiswal S et al., N Engl J Med, 2014). Despite these important clinical associations, the mechanisms by which DNMT3A mutations contribute to malignant transformation have not been well-defined. Dnmt3a-knockout (KO)mouse hematopoietic stem cells (HSCs) preferentially self-renew rather than undergo differentiation, leading to their accumulation in the bone marrow (Challen GA et al., Nat Genet, 2011). DNMT3A loss has also been shown to drive hypomethylation and subsequent activation of leukemia-related genes (Lu R et al., Cancer Cell, 2016; Yang L et al., Cancer Cell, 2016). However, these findings have not been recapitulated using human tissue. The goals of this study were thus to determine the transcriptional and biological effects of DNMT3A mutations which contribute towards malignant transformation in human cells.Methods:To elucidate the effects of DNMT3A mutation, we introduced DNMT3A frameshift mutations into K562 cells using the CRISPR/Cas9 gene-editing system. We then performed various functional and genomic assays to better elucidate effects of DNMT3A loss.Results and Discussion:We successfully created 4 DNMT3A-KO K562 clones and 1 clone containing a mutation that produces an altered DNMT3A protein with an intact catalytic domain (DNMT3A-alt). We first assessed effects of DNMT3A loss on cell growth and apoptosis. DNMT3A-KO clones exhibited impaired growth compared to wild-type (WT) cells. DNMT3A-KO clones also displayed significantly increased apoptotic activity after exposure to 5-fluorouracil (5-FU). The DNMT3A-alt clone had similar growth and apoptotic activity to WT cells. We examined how DNMT3A loss impacted differentiation using phorbal 12-myristate 13-acetate (PMA), known to induce megakaryocytic differentiation of K562 cells. After overnight exposure to PMA, DNMT3A-KO clones exhibited less CD61 expression, a marker of megakaryocytic differentiation, than did WT cells. Again, the differentiation of the DNMT3A-alt clonewas comparable to WT. Finally, we performed karyotype analysis to elucidate a potential role of DNMT3A in maintaining genomic integrity. Surprisingly, DNMT3A-KO clones exhibited profound cytogenetic variability and genomic instability compared to WT, with most DNMT3A-KO clones containing dicentric chromosomes and ring forms in multiple spreads (Figure 1). The DNMT3A-alt clone had a karyotype identical to WT. CRISPR/Cas9-edited K562 clones without DNMT3A mutation (transfected WT or tWT) also had identical karyotypes to WT K562. TA cloning and mRNA sequencing were employed to elucidate whether loss of DNMT3A would lead to transcriptome instability. DNMT3A-KOand DNMT3A-altclones exhibited distorted splicing patterns, while tWT cell lines were comparable to WT. To further assess the effect of DNMT3A ablation on genomic integrity, we examined DNA-damage responses by measuring DNA double-stranded breaks (DSBs) after treatment with 5-FU. DNMT3A-KO clones were significantly more susceptible to DNA damage than were WT cells, while the DNMT3A-alt clone exhibited more DNA DSBs compared to WT only at high concentrations of 5-FU.Conclusion:CRISPR/Cas9-mediated DNMT3A-KO K562 cells may be used to model effects of DNMT3A mutations in human cells. Consistent with previous reports, our data suggest that DNMT3A is involved in the differentiation of multipotent progenitors. Novel to this approach, our findings implicate induction of genomic instability as a mechanism by which DNMT3A mutations might predispose to malignancy. [Display omitted] DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Townsley:Novartis: Research Funding. Young:GSK/Novartis: Research Funding.

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level. We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination. This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain. Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells.

Hematopoietic Stem Cells and Multipotent Progenit c-Kit Receptor: Indication of an Alternative c-Kit Signaling Pathway.

The SCF receptor c-kit plays an important role in the maintenance and differentiation of HSC and multipotent progenitors (MPPs). A new truncated, intracellular form of c-kit receptor, called truncated c-kit (tr-kit), was found first in murine male germ cells. Tr-kit is encoded by a 3.2 kb alternative transcript, which originates in the intron 16 of c-kit gene, and contains a unique 415 bp long 5′ UTR and 36 bp long start of the coding sequence. Besides the extracellular, trans-and juxta-membrane region, the 202 aa long tr-kit protein (Mw. 30 kDa) lacks the ATP binding part of the kinase domain, as well as the hydrophilic kinase insert region. Instead, tr-kit contains a unique 12 aa long hydrophobic region, which is in frame with the 190 aa long C-terminal part of c-kit protein, containing the phosphotransferase domain and C-terminal tail, relevant for interaction with PLCγ. Recently, we have detected tr-kit transcript and protein in murine HSC-like cell line EML, which can differentiate into erythroid, myeloid and lymphoid lineages. Moreover, Western analysis with α-c-kit [pY936] phosphospecific Ab has shown that in EML cells tr-kit is phosphorylated at the C-terminal tyrosine Y936, important for interaction with PLCγ. Thus, we have examined tr-kit expression in FACS-purified murine bone marrow (BM) cell populations highly enriched for long-term and short-term repopulating HSC, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, the tr-kit transcript was detected only in the Lin−Sca-1+c-kit+ Flk2−, Lin−Sca-1+c-kit+ Flk2+ and Lin−Sca-1+c-kit+ BM cell populations, highly enriched for LTR-HSC, STR-HSC and MPPs. On the other hand, the tr-kit transcript was absent inmore heterogeneous Lin− c-kit+ Sca-1−, Lin−Sca-1+ and Lin− Sca-1− BM cells, in which HSC are either present at a low frequency or are absent altogether, andLin+ BM cells, DN thymocytes, CD4 and CD8 T cells, pro-B and pre-B cells, monocytes, macrophages and erythroblasts.To analyze tr-kit expression during differentiation of MPPs into myelo-erythroid lineages, EML cells were cultured in the presence of Epo, GM-CSF, and G-CSF for 72 and 96 hours. The levels of tr-kit transcript and protein were analyzed by quantitative real-time RT-PCR and Western, and the myelo-erythroid differentiation of EML cells was monitored by expression of β-globin and lactoferrin. These experiments have shown that tr-kit transcription and protein expression are quickly down-regulated during myelo-erythroid differentiation of EML cells. More importantly, increased levels of tr-kit protein are associated with SCF-independent maintenance and attenuated differentiation of EML cells, and SCF-independent activation of the full-length c-kit receptor. Together with preferential expression of tr-kit in HSC and MPPs, which also express c-kit, these data suggest that HSC and MPPs could be utilizing distinct SCF-dependent and SCF-independent c-kit signaling. In a proposed alternative model of c-kit function, the maintenance of HSC and MPPs could be mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. Using newly generated tr-kit-specific Ab we are studying interactions of tr-kit with c-kit, PLCγ, and other components of c-kit signaling pathway, and are also analyzing the impact of tr-kit knockdown and over-expression on maintenance and differentiation of EML cells and primary HSC.

Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes.

In this paper, we report analysis of differentiation in human hemopoietic colonies derived from a single cell. Cord blood mononulear cells and panned My-10 antigen-positive bone marrow and cord blood cells were plated in methylcellulose medium containing erythropoietin and conditioned medium. Initially, we performed mapping studies to identify candidate colony-forming cells. Subsequently, using a micromanipulator, we transferred single cells individually to 35-mm dishes for analysis of colony formation. Cellular composition of the colony was determined by identifying all of the cells in the May-Grunwald-Giemsa stained preparation. Of 150 single candidate cells replated, 63 produced colonies. The incidences of single lineage colonies included 19 erythroid, 17 monocyte-macrophage, and 9 eosinophil colonies. There were 18 mixed hemopoietic colonies consisting of cells in two, three, four, and five lineages in varying combinations. In some instances, we noted the predominance of one lineage and the presence of very small populations of cells in a second or third lineage. These results provide evidence for the single-cell origin of human multilineage hemopoietic colonies, and are consistent with the stochastic model of stem cell differentiation in man. They also indicate that restriction of the proliferative potential of committed progenitors is a stochastic process.