Prospective Isolation Research Articles

CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies

Cell-surface markers for prospective isolation of stem cells from human skeletal muscle have been difficult to identify. Such markers would be powerful tools for studying satellite cell function during homeostasis and in pathogenesis of diseases such as muscular dystrophies. In this study, we show that the tetraspanin KAI/CD82 is an excellent marker for prospectively isolating stem cells from human fetal and adult skeletal muscle. Human CD82+ muscle cells robustly engraft into a mouse model of muscular dystrophy. shRNA knockdown of CD82 in myogenic cells reduces myoblast proliferation, suggesting it is functionally involved in muscle homeostasis. CD82 physically interacts with alpha7beta1 integrin (α7β1-ITG) and with α-sarcoglycan, a member of the Dystrophin-Associated Glycoprotein Complex (DAPC), both of which have been linked to muscular dystrophies. Consistently, CD82 expression is decreased in Duchenne muscular dystrophy patients. Together, these findings suggest that CD82 function may be important for muscle stem cell function in muscular disorders.

Identification of leukemia enriched plasma membrane proteins allows prospective isolation and analysis of distinct subclones providing new opportunities for diagnosis and targeting of acute myeloid leukemia

Identification of leukemia enriched plasma membrane proteins allows prospective isolation and analysis of distinct subclones providing new opportunities for diagnosis and targeting of acute myeloid leukemia

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies.

Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application.

Identification and Characterization of Human Endometrial Mesenchymal Stem/Stromal Cells and Their Potential for Cellular Therapy.

SummaryHuman endometrium is a highly regenerative tissue, undergoing more than 400 cycles of proliferation, differentiation, and shedding during a woman's reproductive life. Adult stem cells, including mesenchymal stem/stromal cells (MSCs), are likely responsible for the immense cellular turnover in human endometrium. The unique properties of MSCs, including high proliferative ability, self-renewal, differentiation to mesodermal lineages, secretion of angiogenic factors, and many other growth-promoting factors make them useful candidates for cellular therapy and tissue engineering. In this review, we summarize the identification and characterization of newly discovered MSCs from the human endometrium: their properties, the surface markers used for their prospective isolation, their perivascular location in the endometrium, and their potential application in cellular therapies. The endometrium, or the lining of uterus, has recently been identified as a new and accessible source of mesenchymal stem cells, which can be obtained without anesthesia. Endometrial mesenchymal stem cells have comparable properties to bone marrow and adipose tissue mesenchymal stem cells. Endometrial mesenchymal stem cells are purified with known and novel perivascular surface markers and are currently under investigation for their potential use in cellular therapy for several clinical conditions with significant burden of disease.

A human MIXL1 green fluorescent protein reporter embryonic stem cell line engineered using TALEN-based genome editing

We have generated a MIXL1-eGFP reporter human embryonic stem cell (hESC) line using TALEN-based genome engineering. This line accurately traces endogenous MIXL1 expression via an eGFP reporter to mesendodermal precursor cells. The utility of the MIXL1-eGFP reporter hESC line lies in the prospective isolation, lineage tracing, and developmental and mechanistic studies of MIXL1+ cell populations.

Correction: RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

[This corrects the article DOI: 10.1371/journal.pgen.1005814.].

Isolation and Characterization of Pericytes from Murine Skeletal Muscle

Pericytes are multipotent stem cells located in the perivascular space throughout the microvasculature. Their ability to differentiate into mesodermal cell types and modulate the local tissue environment through paracrine signaling makes pericytes a novel therapeutic target, but their prospective isolation and identification preclude their widespread use in therapeutic research. PURPOSE: To use novel flow cytometry methods to isolate and characterize pericytes from murine skeletal muscle. METHODS: Gastrocnemius and soleus complexes were dissected from CD1 mice, aged 3 months. Muscles were digested using collagenase into a single cell suspension. Cells were filtered through 70 um and 40 um mesh then resuspended in staining medium. Cells were stained at 4°C for 20 minutes with the following antibodies: CD45-BB515 (1:400), CD34-BV421 (1:100), CD56-PE (1:100) and CD146-Alexa647 (1:100). Fluorescence activated cell sorting (FACS) was used to sort CD45-CD56-CD34-CD146+ pericytes using appropriate unstained, single stained, and fluorescence minus one controls. P0 cells were plated on 0.2% gelatin coated plates in EBM-2 complete medium. At 70% confluence, P0 cells were split 1:1 on polystyrene tissue culture plates, and split 1:2 thereafter. Flow cytometry was used to determine the expression of pericyte markers (NG2 and PDGFβ), mesenchymal cell markers (CD73, CD90, and CD105), endothelial (CD31) and hematopoietic cells (CD45) markers on P5 cells. Characterization data were analyzed for the percentage of positive cells and median fluorescent intensity relative to unstained control (nMFI). RESULTS: Pericytes were positive for the pericyte marker CD146 (15.5%, nMFI 102.4), weakly positive for PDGFRβ (10.8%, nMFI 7.5), and weakly positive for NG2 (7.0%, nMFI 6.9). Cultured pericytes were positive for mesenchymal stem cell markers CD73 (52.9%, nMFI 56.9), and CD105 (35.3%, nMFI 9.5), but negative for CD90 (4.9%, nMFI 6.3). Pericytes were negative for the endothelial marker CD31 (2.1%, nMFI 4.4) and hematopoietic marker CD45 (5.4%, nMFI 7.9). CONCLUSIONS: FACS sorting for CD45-CD56-CD34-CD146+ cells yields murine pericytes that can be reliably isolated, definitively characterized, and may be suitable for in vivo and in vitro experiments to test the efficacy of pericyte cell therapy for various diseases.

Gene Expression Profiling Supports the Neural Crest Origin of Adult Rodent Carotid Body Stem Cells and Identifies CD10 as a Marker for Mesectoderm-Committed Progenitors.

Neural stem cells (NSCs) are promising tools for understanding nervous system plasticity and repair, but their use is hampered by the lack of markers suitable for their prospective isolation and characterization. The carotid body (CB) contains a population of peripheral NSCs, which support organ growth during acclimatization to hypoxia. We have set up CB neurosphere (NS) cultures enriched in differentiated neuronal (glomus) cells versus undifferentiated progenitors to investigate molecular hallmarks of cell classes within the CB stem cell (CBSC) niche. Microarray gene expression analysis in NS is compatible with CBSCs being neural crest derived-multipotent progenitor cells able to sustain CB growth upon exposure to hypoxia. Moreover, we have identified CD10 as a marker suitable for isolation of a population of CB mesectoderm-committed progenitor cells. CD10 + cells are resting in normoxia, and during hypoxia they are activated to proliferate and to eventually complete maturation into mesectodermal cells, thus participating in the angiogenesis necessary for CB growth. Our results shed light into the molecular and cellular mechanisms involved in CBSC fate choice, favoring a potential use of these cells for cell therapy. Stem Cells 2016;34:1637-1650.

Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.

The hematopoietic stem cell (HSC) is arguably the most extensively characterized tissue stem cell. Since its identification by prospective isolation1, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology including, self-renewal2–4, differentiation, aging, niche5, and diversity6–8. Here we demonstrate by unbiased multi-step screening, identification of a single gene, Hoxb5 (homeobox B5 also known as Hox-2.1), whose expression in the bone marrow (BM) is limited to the long-term HSC (LT-HSC) in mice. Utilizing a single-color tri-mCherry reporter mouse driven by endogenous Hoxb5 regulation, only the Hoxb5-positive HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients, and critically, in secondary recipients. Only 7–35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse BM, we show that >94% of LT-HSC (Hoxb5+) are directly attached to VE-cadherin-positive cells, implicating a perivascular space as a near homogenous localization of the LT-HSC.

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.

CD146/MCAM defines functionality of human bone marrow stromal stem cell populations.

BackgroundIdentification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC population.MethodsUsing flow cytometry and cell sorting, we isolated two distinct hMSC-CD146+ and hMSC-CD146− cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high-content analysis and additionally for their ability to differentiate toward osteogenesis in vitro and form bone in vivo, and their migrational ability in vivo and in vitro was investigated.ResultsIn vitro, the two cell populations exhibited similar growth rate and differentiation capacity to osteoblasts and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146+ and hMSC-CD146− cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146− cells (12.6 % versus 8.1 %) and bone marrow elements enriched in implants containing hMSC-CD146+ cells (0.5 % versus 0.05 %). hMSC-CD146+ cells exhibited greater chemotactic attraction in a transwell migration assay and, when injected intravenously into immune-deficient mice following closed femoral fracture, exhibited wider tissue distribution and significantly increased migration ability as demonstrated by bioluminescence imaging.ConclusionOur studies demonstrate that CD146 defines a subpopulation of hMSCs capable of bone formation and in vivo trans-endothelial migration and thus represents a population of hMSCs suitable for use in clinical protocols of bone tissue regeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0266-z) contains supplementary material, which is available to authorized users.

FACS Fractionation and Differentiation of Skeletal-Muscle Resident Multipotent Tie2+ Progenitors.

The skeletal muscle niche is complex and heterogeneous. Over the past few decades, various groups have reported the existence of multiple adult stem cell populations within this environment. Techniques commonly used to identify and assess the differentiation capacities of these cellular fractions, oftentimes rare populations, include the use of lineage tracers, immunofluorescence and histochemistry, flow cytometry, gene expression assays, and phenotypic analysis in culture or in vivo. In 2012, our lab identified and characterized a skeletal-muscle resident Tie2+ progenitor that exhibits adipogenic, chondrogenic, and osteogenic differentiation potentials (Wosczyna et al., J Bone Miner Res 27:1004-1017, 2012). This Tie2+ progenitor also expresses the markers PDGFRα and Sca-1 which in turn label a population of muscle-resident fibro/adipogenic progenitors (FAPs) (Joe et al., Nat Cell Biol 12:153-163, 2010; Uezumi et al., Nat Cell Biol 12:143-152, 2010), suggesting similar identities or overlap in the two mesenchymal progenitor populations. Our study demonstrated that these Tie2-expressing mesenchymal progenitors contribute robustly to BMP-induced heterotopic ossification (HO) in mice, and therefore could represent a key cellular target for therapeutic intervention in HO treatment (Wosczyna et al., J Bone Miner Res 27:1004-1017, 2012). In this chapter, we provide a detailed description of our updated fluorescence-activated cell sorting (FACS) strategy and describe cell culture methods for differentiation of Tie2+ progenitors to adipogenic and osteogenic fates. This strategy is easily adaptable for the prospective isolation of other rare subpopulations resident in skeletal muscle.

A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells.

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knock-in reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this ‘imprinting reporter mouse’ can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals the role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease.

Profiling Specific Inner Ear Cell Types Using Cell Sorting Techniques.

Studies of specific tissue cell types are becoming increasingly important in advancing our understanding of cell biology and gene and protein expression. Prospective isolation of specific cell types is a powerful technique as it facilitates such investigations, allowing for analysis and characterization of individual cell populations. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and limited cell markers. In this chapter, we describe methods for selectively labeling and isolating different inner ear cell types from the neonatal mouse cochlea using fluorescence-activated cell sorting.

Prospective isolation of resident adult human mesenchymal stem cell population from multiple organs.

Mesenchymal stem/stromal cells (MSCs) have the potential to form colonies in culture and reside in adult tissues. Because MSCs have been defined using cells cultured in vitro, discrepancies have arisen between studies concerning their properties. There are also differences between populations obtained using different isolation methods. This review article focuses on recent developments in the identification of novel MSC markers for the in vivo localization and prospective isolation of human MSCs. The prospective isolation method described in this study represents an important strategy for the isolation of MSCs in a short period of time, and may find applications for regenerative medicine. Purified MSCs can be tailored according to their intended clinical therapeutic applications. Lineage tracing methods define the MSC phenotype and can be used to investigate the physiological roles of MSCs in vivo. These findings may facilitate the development of effective stem cell treatments.

Systematic Evaluation of Preclinical Genetically Determined Vs. "Stochastic"/Transient Resistance to High-Dose Melphalan Treatment in Multiple Myeloma

Myeloablative high-dose melphalan (HDM) followed by autologous stem cell transplantation (ASCT) remains one of the cornerstones of multiple myeloma (MM) treatment. Conceptually, HDM is based on the notion that a single melphalan dose (typically 200 mg/m2) exceeding the myelotoxicity threshold is associated with a steep increase in the dose response curve and higher degree of MM cell killing compared to fractionated delivery (without the need for hematopoietic stem cell support) of the same cumulative dose. Despite its importance in the therapeutic management of MM, HDM-ASCT is not considered a curative procedure, presumably because chemoresistant subpopulations of MM cells survive HDM and lead to eventual relapse. Elucidating the molecular mechanisms responsible for resistance to HDM in MM could have major implications for the identification of patients with the highest probability of major clinical benefit from this procedure. However, these resistance mechanisms remain incompletely understood. To address this void in the field, we examined whether an open-ended unbiased genome-wide functional characterization of MM cells could uncover genes associated with resistance to HDM and quantify the degree to which other, non-genetically determined, mechanisms of resistance contribute to this process. Specifically, we used the human myeloma cell line MM1.S, transduced with lentiviral construct for the Cas9 nuclease and with pooled lentiviral particles of the GeCKO library (Shalem et al., 2014), which consists of 2 pooled single guide RNA (sgRNA) sub-libraries (~120,000 sgRNAs; targeting ~19,000 genes and ~1800 miRNAs). Using this CRISPR/Cas9-based approach to mutagenize and cause loss of function of the genes recognized by the respective sgRNAs, we sought to facilitate the prospective isolation of MM cells resistant to HDM. To better simulate the exposure of MM cells to HDM in the autologous transplant setting, we adapted our in vitro treatment to include an initial dose of 25 µM of melphalan and sequential one-hour interval partial wash-outs, to achieve gradual reduction of melphalan concentrations, consistent with the pharmacokinetic profile observed in the clinical setting (Nath et al., 2010). We repeated twice the genome-wide knock-out screens, with biological replicates on both the transduction and cell culture level (maintaining a coverage of at least 1000 cells per individual sgRNA). While the in vitro simulation of clinical HDM with our wash-out regimen achieved >99% estimated reduction in MM cell viability in both screens, viable cells re-emerged in cultures from all biological and technical replicates. HDM-resistant cells were processed to quantify their sgRNA enrichment or depletion, using next generation sequencing, and also characterize the sensitivity of these MM cells to repeat exposure to HDM concentrations consistent with the genome-wide CRISPR screens. We observed that, despite having survived a previous round of exposure to HDM, the majority (>90%) of these sgRNA-transduced cells were again highly responsive to repeat treatment with HDM. A similar result was also obtained with MM1.S cells which had not been previously transduced with the sgRNA sub-libraries. While ongoing investigation in our lab addresses these findings on additional MM cell lines, these observations on a well-established MM cell line model of melphalan responsiveness raises the intriguing possibility that genetically-determined forms of HDM resistance, conferred after an unbiased genome scale evaluation through the CRISPR/Cas9-editing methodology, may only represent a small fraction of the MM cells which survive a round of HDM treatment, and that other non-genetically determined mechanisms may mediate the principal mode of resistance, even in the cell autonomous context of our experiments. In turn, this suggests that future efforts to individualize the administration of HDM-ASCT in patients and their post-ASCT monitoring should not rely exclusively on genetic markers identified and validated from preclinical experimentation with repeated rounds of MM cell exposure to the equivalent of myelotoxic melphalan concentrations. Instead, renewed emphasis is warranted on identification of functional markers correlating with the ability of MM cells to develop transient resistance to HDM, even in the absence of constitutive genetically-determined mechanisms of resistance. DisclosuresMitsiades:Novartis: Research Funding; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Definitive Erythro-Myeloid Progenitors (EMPs) Emerge in the Myb-/- Embryo and Retain the Capacity to Differentiate into Macrophages

In the adult, the proto-oncogene Myb critically regulates both the maintenance of hematopoiesis and the differentiation of several hematopoietic lineages. Myb-/- mouse embryos die by embryonic day (E) 15 with severe anemia due to the absence of definitive erythropoiesis (Mucenski et al., Cell, 1991). Similarly, zebrafish embryos lacking myb do not express adult globin genes, have a reduction in other mature hematopoietic lineages by 48 hours post fertilization, and maintain a bloodless phenotype through adulthood (Soza-Ried et al., PNAS, 2010). These and other data have led to the concept that Myb-/- embryos entirely lack definitive hematopoiesis.In both mouse and zebrafish embryos, the first definitive hematopoietic potential arises as a hematopoietic stem cell (HSC)-independent wave of erythro-myeloid progenitors (EMPs). EMPs emerge in the murine yolk sac beginning at E8.25, partially overlapping with an earlier wave of primitive hematopoietic progenitors. We previously demonstrated that EMPs are multipotent progenitors, and are the major source of definitive erythroid potential in the early fetal liver, prior to the colonization of adult-repopulating HSCs (McGrath et al., Blood, 2011). Recently, we identified a unique cell surface phenotype that facilitates the prospective isolation of murine definitive EMPs, distinguishing them from primitive hematopoietic progenitors and maturing populations of megakaryocytes and macrophages in the yolk sac (McGrath et al., Cell Reports, 2015). We detected expression of Myb in sorted EMPs, suggesting that Myb may regulate the emergence and/or differentiation of EMPs. We tested this hypothesis by assessing the emergence, hematopoietic potential and expansion capacity of EMPs, compared with other maturing primitive hematopoietic lineages, in Myb-/- mouse embryos. Consistent with the proposed Myb-independence of the earlier wave of primitive progenitors, we observed normal numbers of maturing macrophages in E9.5 Myb-/- yolk sacs. Interestingly, E9.5 Myb-/- yolk sacs also contained normal numbers of immunophenotypic EMPs. These EMPs were present in hemogenic endothelial-derived clusters expressing Runx1, similar to littermate controls, suggesting that Myb is dispensable for EMP emergence from hemogenic endothelium.We next assessed the differentiation capability of Myb-/- EMPs in vitro. E9.5 Myb-/- yolk sacs lacked high proliferative colony-forming potential (HPP-CFC), a hallmark of immature definitive hematopoietic progenitors. In addition, both definitive erythroid and granulocyte colony-forming potential were absent in methylcellulose cultures of sorted Myb-/- EMPs, in contrast to littermate controls. Surprisingly, however, sorted Myb-/- EMPs gave rise to macrophage progenitors in colony-forming assays, and CD11b+ F4/80+ macrophages in differentiation cultures. These data indicate that Myb is not required for the differentiation of primary definitive EMPs into macrophages. Analysis of Myb-/- fetal liversalso confirmed the presence of F4/80+ macrophages. While these fetal liver macrophages have been previously proposed to be of primitive hematopoietic origin, our data raise the possibility that they may also be derived from EMPs.Further analysis of in vitro differentiation cultures demonstrated an inability of sorted Myb-/- EMPs to proliferate when compared with normal littermates, although these cultures still generated small numbers of macrophages. It is not yet clear whether this reduction in proliferation is due solely to the loss of differentiation of multiple hematopoietic lineages, or is also due to defective maintenance or expansion of EMPs. However, consistent with a role for Myb in continued emergence and/or expansion of EMPs, we observed a reduction in the total number of EMPs by E10.5 in yolk sacs of Myb-/- embryos compared with normal littermates. Taken together, these data indicate that Myb is a critical regulator not only of HSCs, but also of HSC-independent definitive hematopoietic progenitors (EMPs). While Myb is dispensable for the initial emergence of EMPs, it is required for their subsequent differentiation into erythroid and granulocyte lineages. Surprisingly, the persistence of EMPs, while reduced, may provide a source of definitive macrophages in Myb-/- embryos. DisclosuresNo relevant conflicts of interest to declare.

Embigin Regulates HSPC Homing and Quiescence and Acts As a Cell Surface Marker for a Niche Factor-Enriched Subset of Osteolineage Cells

Background. Discovery of niche-derived HSPC regulators is critical for further development of novel therapeutic approaches to promote HSPC regeneration. We have previously reported a proximity-based approach to the study of the bone marrow niche, which is based on the transcriptional comparison of osteolineage cells (OLCs) located close (proximal OLCs) or further away from transplanted HSPC (Blood 2014;124: 773), which lead to identification of IL18 as a quiescence regulator of early progenitors. We now report the results of functional validation for another molecule - Embigin - identified through this strategy as a hematopoietic regulator.Results. Embigin is a cell adhesion molecule with poorly characterized function. Our analysis showed that Embigin expression in proximal OLCs was significantly higher. Since the genetic tools for this molecule do not exist, we used a neutralizing antibody against Embigin to investigate the effect of Embigin blockade on primitive hematopoietic cells. We found that injection of anti-Embigin resulted in mobilization of myeloid progenitors and colony-forming cells (CFC) into the blood. In contrast, Embigin blockade was associated with a homing defect when LKS cells [known to express Embigin] were either pre-incubated with anti-Embigin antibody or injected into anti-Embigin pretreated animals, overall suggesting that Embigin regulates retention and localization of primitive hematopoietic cells in the bone marrow. Moreover, animals treated with anti-Embigin antibody had a higher frequency and proliferative activity of primitive hematopoietic cells, as demonstrated by cell cycle and BrdU incorporation studies and an increased CFC, consistent with Embigin-mediated localization also affecting HSPC cell cycling. Bone marrow from anti-Embigin treated mice reconstituted poorly when competitively transplanted with untreated animal marrow into irradiated recipients, likely due to the impaired homing and increased cell cycling. Finally, pre-treating irradiated recipients with anti-Embigin resulted in increased proliferation of transplanted WT LKS cells. Collectively, these data are consistent with Embigin serving as a regulator of HSPC localization and quiescence.Given a functional role of Embigin in the bone marrow niche and an overlapping pattern of expression with VCAM1, a known niche-derived HSPC regulator, in proximal OLCs we explored the use of Embigin in conjunction with VCAM1 as cell surface markers for prospective isolation of niche factor-enriched OLC subset by flow cytometry. Using this strategy, we were able to purify a rare OLC subset of CD45- Ter119-V CAM-1+E mbigin+ cells (termed VE cells), which were enriched for niche factor expression as compared to their non-VE counterparts. VE cells were transcriptionally distinct from other, previously defined niche subsets such as nestin-GFPdim mesenchymal stem cells, nestin-GFPbright pericytes and N-cadherin-positive osteoblastic cells. In particular, VE cells expressed higher levels of most niche factors than nestin-GFPbright cells - a cell population recently characterized as regulating HSPC quiescence. Interestingly, the expression profile of VE cells from animals transplanted with LT-HSCs and those which were irradiated but injected with saline alone demonstrated upregulation of cell adhesion molecules in the LT-HSC-injected group, suggesting that VE cells are involved in bidirectional communication within the niche.To investigate a functional role of VE cells, we performed co-culture experiments in which we compared the effect of VE and non-VE cells on HSPC proliferation and engraftment. We found that in the presence of VE cells, HSPCs proliferated at slower rate and generated a lower hematopoietic colony number, consistent with quiescence-inducing effect. Upon transplantation, VE-cultured HSPCs generated a higher level of chimerism compared to those cultured on non-VE layer, indicating a superior ability of VE cells to support engraftment and reconstitution properties of HSPC during the in vitro culture.Conclusion. Our work defines Embgin as a previously unrecognized hematopoietic regulator and a cell surface marker for a niche factor-enriched subset of the osteolineage cells which regulates HSPC quiescence. Pharmacological blockade of Embigin signaling may serve as a potential therapeutic tool to enhance hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.