Emerging single-cell tools are primed to reveal functional and molecular heterogeneity in malignant hematopoietic stem cells.

PURPOSE OF REVIEW: The recent emergence of single-cell technologies has permitted unprecedented insight into the molecular drivers of fate choice in blood stem and progenitor cells. This review gives a broad overview of current efforts to understand the molecular regulators of malignant hematopoietic stem cells (HSCs) at the single-cell level. RECENT FINDINGS: The large-scale adoption of single-cell approaches has allowed extensive description of the transcriptional profiles and functional properties of single HSCs. These techniques are now beginning to be applied to malignant HSCs isolated directly from patients or from mouse models of malignancy. However, these studies have generally struggled to pinpoint the functional regulators of malignant characteristics, since malignant HSCs often differ in more than one property when compared with normal HSCs. Moreover, both normal and malignant populations are complicated by HSC heterogeneity. SUMMARY: Despite the existence of single-cell gene expression profiling tools, relatively few publications have emerged. Here, we review these studies from recent years with a specific focus on those undertaking single-cell measurements in malignant stem and progenitor cells. We anticipate this to be the tip of the iceberg, expecting the next 2-3 years to produce datasets that will facilitate a much broader understanding of malignant HSCs

Ceramic identity contributes to mechanical properties and osteoblast behavior on macroporous composite scaffolds.

Implants formed of metals, bioceramics, or polymers may provide an alternative to autografts for treating large bone defects. However, limitations to each material motivate the examination of composites to capitalize on the beneficial aspects of individual components and to address the need for conferring bioactive behavior to the polymer matrix. We hypothesized that the inclusion of different bioceramics in a ceramic-polymer composite would alter the physical properties of the implant and the cellular osteogenic response. To test this, composite scaffolds formed from poly(lactide-co-glycolide) (PLG) and either hydroxyapatite (HA), β-tricalcium phosphate (TCP), or bioactive glass (Bioglass 45S®, BG) were fabricated, and the physical properties of each scaffold were examined. We quantified cell proliferation by DNA content, osteogenic response of human osteoblasts (NHOsts) to composite scaffolds by alkaline phosphatase (ALP) activity, and changes in gene expression by qPCR. Compared to BG-PLG scaffolds, HA-PLG and TCP-PLG composite scaffolds possessed greater compressive moduli. NHOsts on BG-PLG substrates exhibited higher ALP activity than those on control, HA-, or TCP-PLG scaffolds after 21 days, and cells on composites exhibited a 3-fold increase in ALP activity between 7 and 21 days versus a minimal increase on control scaffolds. Compared to cells on PLG controls, RUNX2 expression in NHOsts on composite scaffolds was lower at both 7 and 21 days, while expression of genes encoding for bone matrix proteins (COL1A1 and SPARC) was higher on BG-PLG scaffolds at both time points. These data demonstrate the importance of selecting a ceramic when fabricating composites applied for bone healing

Hematopoietic stem cells depend on HIM and HER

After a series of pioneering experiments on hematopoietic stem cell (HSC) function, including early evidence of heterogeneous stem cell behavior, Jim Till, Ernest McCulloch, and Lou Simminovitch put forward a stochastic model for stem cell proliferation. In contrast to the alternative model, called the "hemopoietic-inductive microenvironment" ("HIM") in which specific microenvironments drove specific and consistent outcomes, they coined the term "hemopoiesis engendered randomly" ("HER"), in which HSCs have intrinsic differences in the cellular state that introduce a probability of potential outcomes. The HIM (extrinsic) and HER (intrinsic) discussion continues nearly 60 years later, but the metaphor also has relevance beyond cellular decision making if one considers the infrastructure and systems supporting the actual scientists who make these advances-a different kind of HER and HIM, but no less important to sort out. This article concludes with some thoughts on how we might achieve a better balance between the HIMs and HERs undertaking the research as well

Clonal heterogeneity as a driver of disease variability in the evolution of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases in which cells of the myelo-erythroid lineage are overproduced and patients are predisposed to leukemic transformation. Hematopoietic stem cells are the suspected disease-initiating cells, and these cells must acquire a clonal advantage relative to nonmutant hematopoietic stem cells to perpetuate disease. In 2005, several groups identified a single gain-of-function point mutation in JAK2 that associated with the majority of MPNs, and subsequent studies have led to a comprehensive understanding of the mutational landscape in MPNs. However, confusion still exists as to how a single genetic aberration can be associated with multiple distinct disease entities. Many explanations have been proposed, including JAK2V617F homozygosity, individual patient heterogeneity, and the differential regulation of downstream JAK2 signaling pathways. Several groups have made knock-in mouse models expressing JAK2V617F and have observed divergent phenotypes, each recapitulating some aspects of disease. Intriguingly, most of these models do not observe a strong hematopoietic stem cell self-renewal advantage compared with wild-type littermate controls, raising the question of how a clonal advantage is established in patients with MPNs. This review summarizes the current molecular understanding of MPNs and the diversity of disease phenotypes and proposes that the increased proliferation induced by JAK2V617F applies a selection pressure on the mutant clone that results in highly diverse clonal evolution in individuals.This is the author's accepted manuscript. The final version of this paper is published in Experimental Hematology here: http://www.exphem.org/article/S0301-472X(14)00622-5/abstract

Targeting tauopathy with engineered tau-degrading intrabodies

BACKGROUND: The accumulation of pathological tau is the main component of neurofibrillary tangles and other tau aggregates in several neurodegenerative diseases, referred to as tauopathies. Recently, immunotherapeutic approaches targeting tau have been demonstrated to be beneficial in decreasing tauopathy in animal models. We previously found that passive immunotherapy with anti-tau antibody to human tau or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system of a mouse model of tauopathy decreased but did not remove all tau-associated pathology. Although these and other studies demonstrate that conventional immunotherapeutic approaches targeting tau can influence tau pathogenesis, the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody. Therefore, we reasoned targeting intracellular tau might be more efficacious in preventing or decreasing tauopathy. METHODS: By utilizing our anti-tau scFv, we generated anti-tau intrabodies for the expression in the cytosol of neurons. To enhance the degradation capacity of conventional intrabodies, we engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations that shuttle intracellular tau for either the proteasome or lysosomal mediated degradation. To evaluate the efficacy in delaying or eliminating tauopathy, we expressed our tau degrading intrabodies or controls in human tau transgenic mice by adeno-associated virus prior to overt tau pathology and after tau deposition. RESULTS: Our results demonstrate, the expression of chimeric anti-tau intrabodies significantly reduce tau protein levels in primary neuronal cultures expression human tau relative to a non-modified anti-tau intrabody. We found the expression of engineered tau-degrading intrabodies destined for proteasomal-mediated degradation are more effective in delaying or eliminating tauopathy than a conventional intrabody in aged human tau transgenic mice. CONCLUSION: This study, harnesses the strength of intrabodies that are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation providing a new immunotherapeutic approach with potentially improved efficacy

Understanding hematopoiesis from a single-cell standpoint.

The cellular diversity of the hematopoietic system has been extensively studied, and a plethora of cell surface markers have been used to discriminate and prospectively purify different blood cell types. However, even within phenotypically identical fractions of hematopoietic stem and progenitor cells or lineage-restricted progenitors, significant functional heterogeneity is observed when single cells are analyzed. To address these challenges, researchers are now using techniques to follow single cells and their progeny to improve our understanding of the underlying functional heterogeneity. On November 19, 2015, Dr. David Kent and Dr. Leïla Perié, two emerging young group leaders, presented their recent efforts to dissect the functional properties of individual cells with a webinar series organized by the International Society for Experimental Hematology. Here, we provide a summary of the presented methods for cell labeling and clonal tracking and discuss how these different techniques have been employed to study hematopoiesis.MRC, Wellcome-Trust, BloodwiseThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.exphem.2016.03.00

Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers