scholarly journals Mass spectrometry analysis of mouse hematopoietic stem cells and their progenitors reveals differential expression within and between proteome and transcriptome throughout adult and aged hematopoiesis

2019 ◽  
Author(s):  
Balyn W. Zaro ◽  
Joseph J. Noh ◽  
Victoria L. Mascetti ◽  
Janos Demeter ◽  
Benson M. George ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stem Cells ◽  
Protein Expression ◽  
Hematopoietic Stem Cells ◽  
Differential Expression ◽  
Cell Types ◽  
Mass Spectrometry Analysis ◽  
Hematopoietic Stem ◽  
Spectrometry Analysis ◽  
Mass Spectrometry Technology

SummaryHematopoietic stem cells (HSCs) are responsible for the generation of blood and immune cells throughout life. They have the unique ability to self-renew and generate more HSCs or differentiate into a progenitor cell in response to cell-intrinsic and -extrinsic stimuli. The balance of HSC fate commitment is critical for a healthy blood supply. Imbalances during hematopoiesis, which are frequent in aging, can result in hematological malignancies and pre-malignancies as well as increase risk of atherosclerosis. Given the importance of HSCs and their progenitors, they have been extensively characterized in genomic and transcriptomic studies. However, an understanding of protein expression within the HSC compartment and more broadly throughout hematopoiesis remains poorly understood, and it has been widely reported that the correlation between mRNA and proteins is more complicated than previously thought. Previous mouse mass spectrometry studies have focused either specifically on stem and the first early progenitor or broadly across mixed populations of stem and progenitor cells, which do not allow for cell-type specific protein resolution across stages of differentiation. Mass cytometry has been employed to characterize transcription factor expression in human HSCs and progenitors but does not apply an unbiased discovery approach. New mass spectrometry technology now allows for deep proteomic coverage with no more than 200 ng of sample input. We report here a proteomics resource characterizing protein expression in mouse adult and aged HSCs, multipotent progenitors and oligopotent progenitors, 12 cell types in total. We validated differential expression by flow cytometry analysis and immunofluorescence staining. Additionally, we investigated the relationship between mRNA and protein levels of individual genes in HSCs compared to progenitors through RNA sequencing studies and identified two proteins that appear to be uniquely regulated in the HSC compartment, Cpin1 and Adnp. In summary, this resource provides proteomic coverage of adult and aged hematopoietic stem cells and their progenitors and reveals changes in protein abundance between cell types, with potential future implications in understanding mechanisms for stem-cell maintenance, niche interactions and fate determination.

scholarly journals Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3758-3779 ◽  
Author(s):  
N Uchida ◽  
HL Aguila ◽  
WH Fleming ◽  
L Jerabek ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Critical Role ◽  
White Blood Cells ◽  
Cell Types ◽  
Dose Dependence ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

Clonal Level Lineage Commitment of Mouse Hematopoietic Stem Cells in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 27-27
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cellular Proliferation ◽  
Population Level ◽  
Cell Types ◽  
Lineage Commitment ◽  
Hematopoietic Stem ◽  
Clonal Level ◽  
Hsc Niche

Abstract Abstract 27 Hematopoietic stem cells (HSCs) sustain the blood and immune systems through a complex differentiation process. This process involves several steps of lineage commitment and forms a paradigm for understanding cellular development, differentiation, and malignancy. While this step-wise differentiation has been extensively studied at the population level, little is known about the lineage commitment of individual HSC clones. The importance of understanding HSC differentiation at the clonal level has been raised by several recent studies suggesting that individual HSCs differentially contribute to various blood cell types and that the aggregate HSC differentiation at the population level is an amalgamation of the diverse lineage commitments of individual HSC clones. The distinct differentiation of individual HSCs may also be accentuated by their regulatory microenvironments, HSC niche. HSC niche may not affect all HSCs in an organism equally, and may instead act directly on resident HSC clones through direct contact or by tuning local cytokine concentrations. Knowledge of HSC clonal level lineage commitment will reveal new insights into HSC regulatory mechanisms and will improve our understanding of aging, immune deficiency, and many hematopoietic disorders involving an unbalanced hematopoietic system. Here, we provide a comprehensive map of in vivo HSC clonal development in mice. The clonal map was derived from the simultaneous tracking of hundreds of individual mouse HSCs in vivo using genetic barcodes. These unique barcodes were delivered into HSCs using a lentiviral vector to obtain a one-to-one mapping between barcodes and HSCs. Barcoded HSCs were then transplanted into recipient mice using standard procedures. Genetic barcodes from donor derived HSCs and their progenies were examined twenty-two weeks after transplantation using high-throughput sequencing. We found that the dominant differentiation of HSC clones is always present in pre-conditioned mice. In these recipients, a small fraction of engrafted HSCs become dominantly abundant at the intermediate progenitor stages, but not at the HSC stage. Thus, clonal dominance is a characteristic of HSC differentiation but not of HSC self-renewal. Additionally, the dominant differentiation of HSC clones exhibits distinct expansion patterns through various stages of hematopoiesis. We provide evidence that observed HSC lineage bias arises from dominant differentiation at distinct lineage commitment steps. In particular, myeloid bias arises from dominant differentiation at the first lineage commitment step from HSC to MPP, whereas lymphoid bias arises from dominant differentiation at the last lineage commitment step from CLP to B cells. We also show that dominant differentiation and lineage bias are interrelated and together delineate discrete HSC lineage commitment pathways. These pathways describe how individual HSC clones produce differential blood quantities and cell types. Multiple clonal differentiation pathways can coexist simultaneously in a single organism, and mutually compensate to sustain overall blood production. Thus, the distinct HSC differentiation characteristics uncovered by clonal analysis are not evident at the population level. We have also identified the lineage commitment profiles of HSC clones belonging to each pathway. These profiles elucidate the cellular proliferation and development of HSCs at the clonal level and demonstrate that distinct modes of HSC regulation exist in vivo. In summary, our in vivo clonal mapping reveals discrete clonal level HSC lineage commitment pathways. We have identified the cellular origins of clonal dominance and lineage bias, which may be the key hematopoietic stages where blood production and balance can be manipulated. These discoveries based on clonal level analysis are unexpected and unobtainable from conventional studies at the population level. Together, they open new avenues of research for studying hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Different Roles of Glycosylphosphatidylinositol in Various Hematopoietic Cells as Revealed by a Mouse Model of Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 2963-2970 ◽  
Author(s):  
Y. Murakami ◽  
T. Kinoshita ◽  
Y. Maeda ◽  
T. Nakano ◽  
H. Kosaka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Cell Clone ◽  
Early Stage ◽  
Fetal Liver ◽  
Cell Types ◽  
Gpi Anchor ◽  
Hematopoietic Stem ◽  
Linked Gene

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have one or a few clones of mutant hematopoietic stem cells defective in glycosylphosphatidylinositol (GPI) synthesis as a result of somatic mutation in the X-linked gene PIG-A. The mutant stem cell clone dominates hematopoiesis by a mechanism that is unclear. To test whether a lack of multiple GPI-anchored proteins results in dysregulation and expansion of stem cells, we generated mice in which GPI-anchor negative cells are present only in the hematopoietic system. We transplanted lethally irradiated mice with female fetal liver cells bearing one allele of the Piga gene disrupted by conditional gene targeting. Because of the X-chromosome inactivation, a significant fraction of the hematopoietic stem cells in fetal livers was GPI-anchor negative. In the transplanted mice, cells of all hematopoietic lineages contained GPI-anchor negative cells. The percentage of GPI-anchor negative cells was much higher in T lymphocytes including immature thymocytes than in other cell types, suggesting a regulatory role for GPI-anchored proteins at an early stage of T-lymphocyte development. However, the proportions of GPI-anchor negative cells in various blood cell lineages were stable over a period of 42 weeks, indicating thatPiga mutation alone does not account for the dominance of the mutant stem cells and that other phenotypic changes are involved in pathogenesis of PNH.

scholarly journals Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression,N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma

2017 ◽  
Vol 16 (10) ◽  
pp. 1705-1717 ◽  
Author(s):  
Carlos A. Morales-Betanzos ◽  
Hyoungjoo Lee ◽  
Paula I. Gonzalez Ericsson ◽  
Justin M. Balko ◽  
Douglas B. Johnson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Expression ◽  
Human Melanoma ◽  
Mass Spectrometry Analysis ◽  
Quantitative Mass Spectrometry ◽  
Spectrometry Analysis ◽  
L1 Protein

scholarly journals The Tao of Hematopoietic Stem Cells: Toward a Unified Theory of Tissue Regeneration

2002 ◽  
Vol 2 ◽  
pp. 983-995 ◽  
Author(s):  
Kevin D. Bunting ◽  
Robert G. Hawley
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Adult Stem Cells ◽  
Developmental Plasticity ◽  
Marrow Cell ◽  
Cell Types ◽  
Therapeutic Benefit ◽  
Hematopoietic Stem ◽  
The Individual

Hematopoietic stem cells (HSCs) are the best studied of the tissue-specific stem cells. By definition, HSCs have long been regarded as restricted to formation of blood cells of both the lymphoid and myeloid lineages. HSCs residing in the bone marrow microenvironment have self-renewal capacity and can repopulate the hematopoietic system of irradiated transplant recipients for the lifetime of the individual. Therefore, HSCs are extremely important targets for gene therapy applications aimed toward the treatment of inherited and acquired blood disorders. However, recent studies have suggested that a subpopulation of HSCs may have the ability to contribute to diverse cell types such as hepatocytes, myocytes, and neuronal cells, especially following induced tissue damage. Preclinical amelioration of liver disease and myocardial infarcts by HSC-enriched bone marrow cell populations raises the possibility that HSC transplants have the potential to provide therapeutic benefit for a wide variety of diseases. These surprising findings contradict the dogma that adult stem cells are developmentally restricted. Extrapolation of these findings to the clinic will be facilitated by prospective identification of the stem cells that possess this developmental plasticity. Furthermore, characterization of the signaling pathways and molecular determinants regulating the remarkable transdifferentiation capacity of these stem cells may provide insight into novel approaches for modulating frequency of differentiative potential.

scholarly journals The CD11a and EPCR marker combination simplifies and improves the purification of mouse hematopoietic stem cells

2017 ◽  
Author(s):  
Alborz Karimzadeh ◽  
Vanessa Scarfone ◽  
Connie Chao ◽  
Karin Grathwohl ◽  
John W. Fathman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gold Standard ◽  
Cell Types ◽  
The Self ◽  
Mouse Bone Marrow ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Marker Combination

AbstractHematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish it from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a, and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, EPCR, can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist’s toolkit improves the purity of and simplifies isolation of HSCs.Significance StatementThe study of hematopoietic stem cells (HSCs) and their purification for transplantation requires a panel of surface markers that can be used to distinguish HSCs from other cell types. The number of markers necessary to identify HSCs continues to grow, making it increasingly difficult to identify HSCs by flow cytometry. In this study, we identified a combination of two surface markers, CD11a and EPCR, to enrich for HSCs in the mouse bone marrow without the need for additional markers. This simplified panel could aid HSC research by reducing the number of markers necessary to identify and isolate HSCs.

Different protein expression of healthy and malignant hematopoietic stem cells

2005 ◽  
Vol 23 (16_suppl) ◽  
pp. 6636-6636
Author(s):  
H. Lannert ◽  
T. Franz ◽  
A. Bathke ◽  
A. Lenze ◽  
R. Hofmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem

Connexin-43 Expression Regulates the Migration of Hematopoietic Stem Cells and Progenitors towards and From Bone Marrow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 562-562
Author(s):  
Daniel Gonzalez-Nieto ◽  
Kyung-Hee Chang ◽  
Anja Koehler ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Connexin 43 ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Hematopoietic Microenvironment ◽  
Deficient Mice

Abstract Abstract 562 In the bone marrow (BM) cavity, the migratory traffic of hematopoietic stem cells and progenitors (HSC/P) from the endosteal niches to circulation and viceversa depends on their response to chemokine gradients and interaction with endothelial and mesenchymal pre-osteoblastic cells located at the endosteal niches, forming the hematopoietic microenvironment (HM). Several lines of evidence have pointed out the possible role of the gap junction-forming protein connexin-43 (Cx43) in the control of stem cell and progenitor migration. Our group previously demonstrated that Cx43 expression in the hematopoietic microenvironment (HM) is critical in the fetal liver and BM hematopoietic regeneration after administration of 5-fluorouracil (5-FU) and other investigators have shown that Cx43 is crucial controlling the migration of neural progenitors along radial glial during brain development. We hypothesized that Cx43 could regulate the bidirectional migration of HSC/P in the BM stroma. Since Cx43 is expressed by mesenchymal cells, endothelial cells and hematopoietic stem cells and progenitors, we decided to analyze the Cx43 contribution in the control of HSC/P migration in cell-specific conditional knock-out mice. To achieve this objective, we have used mice that were selectively deficient for Cx43 in the osteoblast/stromal cells (Collagen 1a-Creflox/flox; O/S-Cx43-deficient), in endothelial cells (Tek-Creflox/flox; E-Cx43-deficient) or in hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). O/S-Cx43-deficient mice have been shown to be a model of osteoblast loss of function (Chung DJ et al., J. Cell. Sci., 2006) and E-Cx43-deficient mice have been shown to be a model of arterial hypotension induced by both increase nitric oxide and angiotensin levels (Liao Y et al, PNAS 2001). Analysis with reporter crossings with Rosa-loxP-Stop-LoxP-LacZ mice showed anatomical specificity of the Cre recombinase expression in different cell types of BM, and western-blot and RT-PCR expression indicated practical abolishment of the expression of Cx43 in each of the specific cell types. First, we analyzed whether there were changes in the levels of circulating progenitors in O/S-, E- or H-Cx43-deficient mice. While H-Cx43-deficient mice did not show any change in the levels of circulating HSC/P, E-Cx43-deficient mice showed a 3.5-fold and 4.7-fold, respectively, increase of circulating CFU-C and competitive repopulating units while maintaining normal repopulation ability of BM HSC. O/S-Cx43-deficient mice showed a 30% reduction in basal conditions which was more accentuated when administered G-CSF (50% reduction on day +6), compared with their WT counterparts. Interestingly, while osteoblast loss-of-function was induced in O/S Cx43-deficient mice, the intramarrow expression levels of CXCL12a/b and mesenchymal progenitor content (CFU-F) were increased (4- and 2-fold, respectively). In correlation with the increased levels of CXCL12, the distance to endosteum of transplanted CFSE+/lin-/c-kit+ BM cells into non-myeloablated O/S-Cx43-deficient mice was dramatically decreased (36.1±4.3 vs 23.2±2.1 mm, p<0.01), suggesting a major change in the cellular composition and chemokinesis within the hematopoietic microenvironment “in vivo”. Interestingly, the 16-hour homing of HSC/P transplanted into lethally irradiated O/S-Cx43KO recipient mice showed a ∼60% reduction and a significantly decreased survival in a limiting-dose transplantation radioprotection assay (50% survival in WT mice vs 0% survival in O/S Cx43-deficient recipients). The homing/engraftment defect of these mice correlated with a reversal of the increased levels of CXCL12 in irradiated BM and a 50% reduction of the migration of WT HSC/P through O/S-Cx43-deficient stroma in response to CXCL12. Altogether, these data indicate that intercellular communication through Cx43 shares distinct functions between the different cell components of the hematopoietic microenvironment, and mediates CXCL12-dependent and CXCL12-independent mechanisms in control of the BM homing and retention of HSC/P. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document