University of Groningen
On the molecular mechanisms of hematopoietic stem cell aging Lazare, Seka Simone
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CHAPTER 5
The Neogenin Receptor in Hematopoiesis
Seka Lazare
1, Johannes Jung
1, Arthur Flohr Svendsen
1, Albertina
Ause-ma
1, Ellen Weersing
1, Gerwin Huls
3, Jan Jacob Schuringa
3, Ronald van
Os
1,2, Leonid Bystrykh
*1, Gerald de Haan
*1,21 European Research Institute for the Biology of Ageing,
2 Mouse Clinic for Cancer and Aging, University Medical Center Groningen, University of Groningen Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands,
3 Department of Experimental Hematology, University Medical Center Groningen, Groningen, The Netherlands
Abstract
Since its discovery the role of Neogenin, a multifunctional receptor implicated in proliferation, differentiation and migration, has been most frequently reported in neuronal development. However, recent studies have hinted that this receptor may play a role in other tissues and processes, from cell-fate decisions of embryonic stem cells to inflammation. We found Neogenin to be one of only two genes most frequently reported as deregulated in hematopoietic stem cell aging. We show here that Neogenin expression is positively correlated with self-renewal potential of murine hematopoietic stem and progenitor cells, and is highly enriched in LT-HSC. By knocking-down Neogenin, we demonstrate for the first time that it is required for functioning of aged HSC, both in vivo and in vitro. In humans, Neogenin is highly expressed on a subset of cord-blood derived HSCs. These Neogeninhi cells have
reduced stem-cell potential when tested in vitro. Overall, our data show that Neogenin expression is able to distinguish HSC subsets with different stem cell potential and that knock-down of Neogenin expression affects proliferation and repopulating ability of HSCs. These results highlight Neogenin as an important receptor for hematopoietic stem cell function and aging.
Introduction
Although many studies have reported deregulation of gene expression with aging of hematopoietic stem cells (HSCs), we have previously shown that there is very little overlap in gene lists between studies. Our previous work, which used a combinatorial analysis of genes most consistently deregulated in individual mice and those reported in previous studies, found that only two genes had been consistently reported as upregulated in HSC aging. These genes, Neo1 and Spdr, have surprisingly never been the focus of hematopoietic stem cell studies.
We decided to focus on Neogenin for multiple reasons. First, it has been previously implicated in differentiation, migration, inflammation and proliferation of certain cell types 1–6, all processes which are thought to be modulated in HSC aging. Secondly, as
a transmembrane receptor, it allowed for the isolation and ligand-induced perturbation of HSCs based on Neogenin expression for further functional investigation. Thirdly, although Neogenin has been associated with LT-HSCs indirectly via gene lists of differentially expressed genes enriched in LT-HSCs and a LT-HSC subpopulation 7–12,
it has never directly been experimentally investigated in this way, and offered some novelty.
The structure of Neogenin is highly similar to the tumor suppressor Deleted in Colorectal Cancer (DCC). Neogenin is an extremely large protein (190kDa) 13, and
contains three structural domains; an extracellular domain consisting of IgG-like loops and fibronectin repeats, a transmembrane domain, and an intracellular domain through which downstream signaling occurs 14 (Figure 1A). The multifunctional role
of Neogenin is facilitated by its ability to bind multiple families of ligands, mainly Netrins and Repulsive Guidance Molecules (RGMs) 1. RGMs and Neogenin are able
to act as co-receptors for BMPs and are thought to enhance BMP signaling in this way 15. Different Neogenin ligands (illustrated in Figure 1B) (even within the same
family) are able to bind distinct sites of the extracellular domain of Neogenin, and this is thought to play a role in the different effects these bindings generate. For example, during axon development, axons demonstrate chemoattraction towards Netrin-1 but chemorepulsion to RGMa 16. Spatially controlled gradients of these two ligands aid
in the precise localization of axons along the spinal cord. Similarly, embryonic stem differentiation has been shown to be delayed by Netrin-1, but accelerated by RGMc 17.
The intracellular domain of Neogenin has been less characterized than its extracellular domain, but has been shown to interact with Focal-adhesion and Src kinase family members, in addition to transcription factors Lim-only Binding Protein 4 (LMO4) and Nuclear factor of activated T Cells (NFATc3) 2,13,18–20 alluding to the ability of
Neogenin to not only activate downstream pathways, but also to activate transcription. Beyond the interacting partners, the intracellular domain has been reported to have transcriptional regulatory activity through translocation to the nucleus 21, and
pro-apoptotic activity brought about by cleavage of its Caspase-3 motif 22,23.
In light of the complexity of Neogenin and its downstream effects, our initial questions were straightforward: We wished to investigate whether Neogenin was expressed in hematopoietic stem and progenitor cells, to validate its upregulation with HSC aging at the RNA and protein level, and to determine if perturbations of Neogenin expression could affect HSC function.
Methods
Mice
C57BL/6, C57BL/6.SJL and W41.SJL were obtained from the Central Animal Facility (CDP) at the University Medical Center Groningen. All experiments were approved by the requisite regulatory authorities.
Mouse HSC and Progenitor Cell Isolation
Bone marrow was isolated from the tibia, femura, pelvis, sternum and spine by crushing, and red blood cells were lysed with erylysis buffer. The white blood cells were stained with a cocktail of antibodies as follows: antibodies detecting lineage (Lin) markers (B220, CD3, Gr-1, Mac-1 and Ter-119) were conjugated with Alexa 700, antibodies detecting c-Kit were conjugated with Phycoerythrin, antibodies detecting Sca-1 were conjugated with Pacific Blue, antibodies detecting CD48 were conjugated with Alexa 647, and antibodies detecting CD150 were conjugated with PeCy7, CD16/42 PeCy7, CD127 Phycoerythrin and CD34 FITC (all antibodies were purchased from Biolegend). These antibodies were used in combination to isolate LT-HSC, ST-HSC and progenitor
populations as detailed below.
LT-HSCs were characterized as Lin-Sca-1+c-Kit+CD48-CD150+, ST-HSCs as Lin-
Sca-1+c-Kit+CD48-CD150- and MPPs as Lin-Sca-1+c-Kit+CD48+CD150-). For isolation of
isolation:
CLP (Lin-CD127+Sca-1loc-Kitlo), CMP (Lin-CD127-Sca-1-c-Kit-CD34+CD16/CD32hi),
GMP (Lin-CD127-Sca-1-c-Kit+CD16/CD32hi), and MEP (Lin-CD127-Sca-1-
c-Kit+CD34-CD16/CD32+).
All cells were isolated on MoFlo Astrios or XDP cell sorters (Beckman Coulter).
!"#$%&'()*++,-./01-2(23/01()4+20&1 51!!! 6(,(07-!17/08(%%9%0/)4+20&1 A !"#$%! &'(')'*& $*+,'-'. -+/0$*+ B
Figure 1 Structure of Neogenin and its
receptors. A. The Neogenin receptor is part of the
immunogloubin family of receptors, consisting of externally of 4 Immunoglobulin-like (IgG-like) residues and 6 Fibronectin III (FnIII) repeats. Neogenin’s intracellular and external domains are separated by a transmembrane domain. The intracellular doman consists of 3 subunits, whose residues have been shown to interact with signaling activators (e.g. Src kinase and Focal adhesion kinase) and transcription factors (e.g NFATc3 and LMO4). B. Neogenin ligands consist of two familys; Netrins and Repulsive Guidance Moclules (RGMs) listed here. Neogenin is also able to enhance Bone-morphogenic signaling in combination of BMPs and RGM proteins.
Human CD34 Cell Isolation
Cord blood was obtained from healthy full-term pregnancies after informed consent in accordance with the Declaration of Helsinki from the obstetrics department at the Isala hospital in Zwolle, the Netherlands. Initially, cord blood volume and cell counts were measured and then diluted 1:1 with PBS 2mM EDTA 0.5% BSA. Maximum 30 ml of diluted cord blood was carefully layered on 15 ml of Lymphoprep™ in a 50ml falcon tube and centrifuged for 20 minutes, 800g, without brakes. Middle layer containing mononuclear cells were harvested and diluted 1:1 with PBS 2mM EDTA 0.5% BSA and then centrifuged for 5 minutes at 800g. Cell pellets were collected and washed with PBS 2mM EDTA 0.5% BSA and centrifuged for 10 minutes at 200g. Immunomagnetic labeling and separation was performed according to the manufacture manual of the CD34 MicroBead Kit, human (Miltenyi Biotec). Cells were either used immediately for experiments or frozen in Cryostor CS10. For isolated of CD34-CD38
-Neo1+ and Neo1- cells, approximately 1x106 CD34+ enriched cord blood cells were
blocked for 30 minutes with 4% BSA, washed and stained with CD34 APC, CD38 PE (BD Biosciences) and rabbit anti-human Neogenin (Santa Cruz) for 30 minutes at 4°C. After washing, cells were stained with 1:100 goat anti-rabbit Alexa 488 (Invitrogen). Cells were isolated on Moflo XDP cell sorter (Beckman Coulter).
For isolation of Bone Marrow CD34+ cells, after achieving informed consent, bone
marrow aspirates were obtained from patients age >60 years (old) who underwent total hip replacement, volunteers age 18 to 35 years (young), and young healthy potential donors for hematopoietic Allogenic Stem Cell Transplantation who underwent BM aspiration as part of a standard medical examination. The protocol for normal bone marrow (NBM) collection was approved by the Institutional Review Board of the University Medical Center Groningen. All participants had normal general health and normal peripheral blood counts and an absence of hematologic disorders. Mononuclear Cell fraction isolation and immunomagnetic separation of CD34+ cells was performed
using Lymphoprep™ and CD34 MicroBead Kit, human (Miltenyi Biotec) respectively according to manufacturer’s instructions.
qPCR Gene Expression Analysis
At least 10,000 LT-HSC, ST-HSC, MPP, CLP, GMP and MEP were sorted directly into lysis buffer and RNA was isolated using the Nucleospin RNA XS Kit (Machery Nagel). RNA quality and quantity was determined (Bioanalyzer, Agilent Technologies). RNA was reverse transcribed into cDNA with the Superscript VILO cDNA Synthesis Kit (Invitrogen). Amplicons for Neogenin and housekeeping gene HPRT were amplified and quantified via qPCR using LightCycler SYBR Green I Master mix and LightCycler 480 Instrument (Roche). Neogenin expression was normalized relative to the housekeeping gene HPRT.
Gene Expression Profiling of Bone marrow CD34+ Cells
Young NBM samples for the microarray analysis came from young healthy volunteers and young potential donors. All older NBM samples came from patients undergoing
total hip replacement.
Total RNA was isolated using the RNeasy mini kit from Qiagen according to the manufacturer’s recommendations. RNA quality was examined using the Agilent 2100 Bioanalyzer (Agilent Technologies). Genome-wide expression analysis was performed on Illumina (Illumina, Inc.) BeadChip Arrays Sentrix Human-6 (46k probesets). Typically, 200 ng of mRNA was used with Illumina Totalprep RNA Amplification Kit (Ambion) and 750 ng of cRNA was used in labeling reactions and hybridization with the arrays according to the manufacturer’s instructions. Data were analyzed using the BeadStudio v3 Gene Expression Module (Illumina, Inc.) and Genespring (Agilent Technologies). In total, we analyzed 11 young (age 18-35) and 22 old (age>65) bone marrow CD34+ samples.
Immunofluorescence Staining
4000-6000 LT-HSC were seeded onto spots of an immunofluorescent adhesion Slide (VWR) and allowed to settle for 20 minutes. Cells were simultaneously fixed and permeabilized with Fixation and Permeabilization Solution (BD Biosciences) for 20 minutes on ice. Cells were then blocked with 4% BSA for 30 minutes at room temperature and stained with 1:100 Mouse Neogenin Biotinylated Antibody (R and D Systems) and alpha tubulin rabbit anti-mouse antibody Alexa-488 (Abcam) overnight at 4°C. Cells were washed three times in 0.1% Tritox-X-100 PBS solution and stained with 1:500 secondary antibody streptavidin Alexa-647 for one hour at 4°C. After washing, coverslips were mounted with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher).
Images were acquired on a Leica Sp8 confocal microscope and quantified on Fiji Image J.
Intracellular Flow Cytometry
LT-HSC were fixed and permeabilized using Fixation and Permeabilization Solution (BD Biosciences) for 20 minutes at 4°C. Cells were washed twice in 0.1% Triton-X-100 and blocked with 4% BSA for 30 minutes at room temperature. Cells were stained with 1:100 Mouse Neogenin Biotinylated Antibody (RnD Systems) for 1 hour at 4°C. Following two washes in 0.1% Triton-X-100, cells were stained with 1:500 secondary antibody streptavidin Alexa-647 for 30 minutes at 4°C. Cells were washed in 0.1% Triton-X-100 and resuspended in 0.2% PBS/BSA for acquisition. Fluorescence was measured on BD FacsCantoII (BD Biosciences) and data analyzed on Flowjo (Flowjo, LLC).
HSC Lentiviral Transduction
Neogenin and SCR shRNA plasmid DNA were purchased from Sigma-Aldrich and subcloned into PLKO.1 mCherry vector (kindly provided by Dr. Hein Schepers, Dept of Hematology, UMCG). For lentivral particle production, HEK293T cells were transfected with 3µg plasmid DNA (Neo1 shRNA or SCR control vectors), 0.7µg
VSV-G envelope vector, and 3µg packaging construct pCMV8.91 in the presence of Fugene HD (Roche). Viral supernatant was collected 24-36 hours later.
For transduction of LT-HSC, purified cells were cultured for 24 hours prior to transduction in Stemspan (StemCell Technologies) supplemented with 10% FCS, and cytokines 300 ng/mL recombinant mouse Stem Cell Factor (Peprotech), 100ng/mL recombinant mouse IL-11 (R&D Systems) and 100ng/mL Flt3 ligand (Amgen). After 24 hours in culture, LT-HSC and viral supernantant were combined in a retronectine (TaKaRa Bio) coated wells and spun for 60 minutes at 400g after which supernatant was removed and cells resuspended in fresh media supplemented with cytokines for a further 24 hours.
Single Cell Colony Assay
Single LT-HSCs transduced with either Neo1 or SCR shRNA (mCherry+) were
sorted into the inner 60-wells of round-bottom 96-well plates and cultured for 14 days in Stemspan (StemCell Technologies) supplemented with 10% FCS, 300ng/ml recombinant mouse Stem Cell Factor (Peprotech), 30ng/ml IL-11 (R&D systems) and 10ng/ml Flt3 ligand (Amgen). Colony sizes were scored at day 14 at 50x magnification using an oculair cross on Zeiss Axiovert 25.
Bone Marrow Transplantation and chimerism analysis
Transduced LT-HSC from B6 donor mice were mixed with W41.SJL competitor whole bone marrow cells and subsequently transplanted, 24 hours post transduction, into lethally irradiated (9Gy) B6.SJL recipients.
For blood chimerism analysis, 250ul of peripheral blood was lysed with erylysis buffer and stained for CD45.2 BUV395, CD3 APC, B220 FITC, Gr-1 A700 and Mac-1 A700 (all from BD Biosciences). Samples were acquired on BD LSRII (BD Biosciences) and analyzed on Flowjo (Flowjo LLC).
LTC-IC Assay
The CAFC and LTC-IC assays were performed as previously described 24,25. Limiting
dilution frequencies were analysed using ELDA 26.
Results
Neogenin is highly expressed in LT-HSC and upregulated in aged Hematopoietic Stem Cells
Our previous global RNA-Seq experiments (Chapter 4) found Neogenin to not only be upregulated in our analysis of gene expression changes in Old HSCs compared to young (Figure 2B) but to be one of two of the most consistently upregulated genes in murine HSC aging. In our investigation of genes upregulated in individual young and old mice compared to previously reported studies using pools of mice, not only was Neogenin upregulated in all old mice, it was also the most reported gene in other studies (Figure 2A).
We first wanted to validate these genome-wide RNA-Seq experiments and demonstrate that Neogenin was upregulated in HSCs at both the RNA and protein level. Indeed, Neogenin RNA expression was significantly upregulated (~5-fold) in old vs young HSCs as measured by qPCR analyses on RNA isolated from old or young HSCs (Figure 2C). To measure protein expression in individual HSCs, we used intracellular flow cytometry (Figure 2D) and confocal microscopy (Figure 2E). Again, Neogenin was significantly upregulated in old HSCs compared to young, however at the single cell level this was not a global upregulation, as many old HSCs had comparable levels to young. Instead there was an emergence of a population of Neogeninhi HSCs in the aged HSC pool (Figure 2D). We did observe a difference in polarity, where Neogenin had a polarized expression in the majority of young HSCs but was more evenly distributed in aged HSCs (polarity observed in 60.7% young HSCs vs. 38.1% aged HSCs).
To gain further insight into the implication of Neogenin upregulation in aged HSCs, we wondered how the expression of Neogenin varied in cell types with different self-renewal and differentiation potentials. For this reason, we measured Neogenin RNA expression among the stem and progenitor compartment, from LT-HSCs with enhanced self-renewal and differentiation capabilities, to cell types with reduced and no self-renewal (ST-HSC and MPPs respectively) and cell types with lineage-restricted differentiation (i.e. Common Lymphoid (CLP), Common Myeloid (CMP), Megakaryocyte Erythrocyte (MEP) and Granulocyte Macrophage (GMP) progenitors). Across the entire hematopoietic tree, Neogenin expression was positively correlated with self-renewal potential. LT-HSC expressed highest levels of Neogenin compared to all other cell types and expression levels decreased drastically from LT-HSC to ST-HSC, and again from ST-HSC to MPP. All committed progenitors expressed negligible levels of Neogenin compared to LT-HSC, with no significant differences between them (Figure 3A).
Knock-down of Neogenin in Aged HSCs Impairs function in vitro and in vivo
As Neogenin expression is upregulated with age, we questioned whether there would be phenotypic consequences of repressing Neogenin in different degrees of Neogenin knock-down. One shRNA (Neo1 shRNA 1) resulted in 25% downregulation of Neogenin whereas the other (Neo1 shRNA 2) resulted in 80% downregulation (Figure 3B). When aged LT-HSCS were lentivirally transduced with these shRNAs and their proliferation tested over 14 days via a single-cell colony assay, we saw a dose-dependent effect of Neogenin knock-down on HSC proliferation. For both shRNAs, knock-down of Neogenin resulted in a reduction of HSC proliferation and this reduction of proliferation was dependent on the level of knock-down, with Neo1 shRNA 2 having the lowest number of HSCs forming colonies and the colonies that did grow were significantly smaller than SCR control or Neo1 shRNA 1 (Figure 3C). To assess the consequences of repressing Neogenin in HSCs to functioning of these cells in vivo, we lentivirally transduced LT-HSC with the most potent Neo1 shRNA
M1 M2 M3 M4 M5 Selp Neo1 Sdpr Vldlr Cysltr2 Ehd3 Pbx3 Mt1 Bcl3 Plscr2 Osmr Ampd3 Klhl4 Enpp5 Gda Plk2 Ramp2 Sult1a1 Cyp26b1 Trim47 Nupr1 Itgb3 Cpne8 Cldn5 Gstm2 Cd38 Lamp2 Arhgap29 Matn4 Plxnb2 Lsr Clec1a 6 0 3 M5 M5 M5 Number of Studies
DAPI NEOGENIN TUBULIN MERGE
OLD YOUNG Receptor Epigenetic Regulator Transcription Factor Development Cell Cycle Histones Upregulated Downregulated Young HSC Old HSC 0 100 200 300 No rm al ize d co un ts p er m illi on R ea ds *** Young HSC Old HSC 0 5 10 15 R el at iv e ne o1 e xp re ss io n * Young Old HSC 0 5.0×1004 1.0×1005 1.5×1005 2.0×1005 2.5×1005 N eo ge ni n **** A B C D E
Figure 2 Neogenin is upregulated in aged HSCs. A.
Combinatorial analysis of top differentially expressed genes in individual aged mice in our data and in other reported studies (including ours). Grey bars indicate number of times a gene has been reported by various studies
8,9,10,11,12 to be differentially expressed in aged HSCs compared to young.
Boxes under individual mice (designated M1-5) represent significant differential expression from average expression in young HSCs. Genes are categorized according to the color coding shown at the bottom of the figure. B. Normalized counts per million (cpm) of Neogenin expression between young and old HSC in our RNA-seq analysis (described in Chapter 4). * denotes significant T-test p value (p<0.05). C. Relative Neo1 mRNA expression as analysed by qPCR normalized on the house-keeping gene HPRT and young expression. * denotes significant T-test p value (p<0.05). D. Neogenin protein expression in young and old HSC measured by intracellular flow cytometry. * denotes significant T-test p value (p<0.05). E. Confocal images of Neogenin expression in young and old HSCs. Scale bar = 5µm.
(shRNA 2) or with a SCR control vector, and competitively transplanted these cells -in vivo into irradiated recipients, using W41.SJL whole bone marrow as competitors. All Neogenin knock-down recipients failed to engraft up to 12 weeks (<1% mCherry+ peripheral blood cells), in contrast to SCR controls which demonstrated robust engraftment (Figure 3D).
Downregulation of Neogenin in Young HSCs has no effect on repopulation
We next sought to investigate whether the observed effect of Neogenin downregulation was specific to aged HSCs. We therefore transduced young LT-HSC (isolated from 4-6 month old mice) with Neo1 shRNA2 and again, transplanted these cells in vivo into irriadiated recipients using W41.SJL whole bone marrow as competitors. Surprisingly, young HSCs in which Neogenin expression was repressed were able to engraft and contribute to blood production and there was no significant difference between young Neo1 knock-down cells and SCR control in vivo in terms of whole blood chimerism (mCherry+ cells) (Figure 3F), although they did show a reduction in proliferation in
vitro (data not shown). Neogenin knock-down HSC displayed some lineage bias in repopulation (Figure 3G), although this was not statistically significant at 20-weeks post transplantation.
Neogenin is expressed on a subset of human Umbilical Cord CD34+CD38- HSCs
and isolated Neohi cells display reduced in vitro functionality
As Neogenin is a transmembrane receptor protein, we sought to isolate HSCs based on their Neogenin expression by Fluorescent Activated Cell sorting (FACS) and investigate the functional difference between Neohi and Neolo cells. The availability
of commercial antibodies for Neogenin is sparse, and the only suitable antibody for FACS provided detectible signal only human Neogenin. Like murine HSCs, Neogenin was also found to be upregulated in human bone-marrow CD34+ Cells (Figure 4A).
Therefore, we sought to investigate functional differences in Neohi and Neolo cells
in human HSCs, using cord blood derived HSCs. Human CD34+CD38- cord blood
cells do express Neogenin and a subset of CD34+CD38- expressed Neogenin to a
comparably high level, compared to CD34- cells (Figure 4B and C).
To determine whether there was any functional difference between CD34+CD38
-Neogeninhi and Neogeninlo cells we isolated these subsets from human umbilical cord
CD34+CD38- cells by FACS and co-cultured them with MS-5 stromal cells in 96-well
plates. At day 35 wells were scored for the presence of hematopoietic cells, which were further categorized into output cell activity (differentiated) or cobblestone-area cell forming colony (primitive). While Neogeninlo cells resulted in a majority of wells
with hematopoietic activity and substantial CAFC activity at day 35, Neogeninhi cells
had much less or no output or CAFC activity (Figure 4D).
To further test HSC activity, we subjected these co-cultured cells at day 35 to the LT-IC assay by subsequent addition of methylcellulose and cultured them for an additional 7 days. Newly emerged colonies were scored to determine LT-IC frequency. Again,
Neogeninhi cells had no/little LT-IC frequency compared to Neogeninlo cells (Figure 4E and 4F). LT-HSC ST-HSC MPP CLP CMP GMP MEP 0.00 0.05 0.10 0.150.2 0.4 0.6 0.8 1.0 Rela tiv e n eo 1 ex pres sio n **** *** *** ****
SCR Neo1 ShRNA 1 Neo1 ShRNA 2
0.0 0.5 1.0 R el at iv e n eo 1 exp res si o n
SCR Neo1 ShRNA 1 Neo1 ShRNA 2
0 12 34 56 shRNA Colon y Siz e 40 12 25 17 11 8 7 30 14 42 20 10 4 92 17 6 4 1 A B C E F G ** ** **** ****
Figure 3 Functional Analysis of Neogenin Repression. A. Neogenin is preferentially
expressed in LT-HSC compared to progenitors. Q-PCR analysis of Neogenin expression in the HSPC compartment in young mice. LT-HSC = long-term hematopoietic stem cells, ST-HSCs = short-term hematopoietic stem cells, MPP = multipotent progenitors, CLP = common lymphoid progenitors, CMP = common myeloid progenitors, GMP = granulocyte-macrophage progenitor, MEP = megakaryocyte– erythroid progenitor. Expression is normalized to housekeeping gene HPRT and on LT-HSC expression. * denotes significant T-test p value (p<0.05). B. Knock-down efficiency of Neo1 shRNA1 and 2
6HFRQGDU\2QO\
&RUG%ORRG&'
Cells
Forward Scatter Height
Side Scatter Height Singlets Forward Scatter Height
Side Scatter Width
PI-82.5 PE PI CD34+CD38 -CD34 -CD38 CD34 Neogenin &RUG%ORRG&' Neogenin Neogenin CD38 CD38 CD38
Neo1lo Neo1hi Neo1lo Neo1hi
No Activity Output Cells CAFC Cord 1 Cord 2 &'&'í6XEVHW CAFC Score 60 28 28 4 48 10 2 21 30 9 &'&'+XPDQ&RUG%ORRG6XEVHW 'D\&$)&6FRUH Neolo Neohi 0 5000 10000 15000 20000 LT -IC per 10 5 HSC CD34+CD38- Subset 0 50 100 150 200 250 !3.0 !2.5 !2.0 !1.5 !1.0 !0.5 0.0
dose (number of cells)
log fr action nonresponding Group 345_Hi Group 345_Lo Group 346_Hi Group 346_Lo A C D E F B
Figure 4 Functional Analysis of Neohi Subsets in CD34+CD38- Cord Blood. A.
Neogenin is upregulated in old human bone-marrow CD34+ cells compared to young. Neogenin
mRNA expression measured using Illumina BeadChip Array. * denotes significance T-test
p-value. B. Gating strategy and Neogenin expression in CD34+CD38- and CD34- Cord Blood
Cells. C. Frequency of Neohi cells in CD34+CD38- and in CD34- cells in 7 individual cord
blood isolates. D. Day 32 Cobblestone Area Forming Cell Analysis of Neohi cells and Neolo
CD34+CD38-. E-F. LTC-IC Frequency of Neohi cells and Neolo CD34+CD38-.
normalized to SCR control as measured by qPCR. C. Day 14 single cell colony sizes of LT-HSC with Neo1 shRNA 1 and 2 and SCR control. Cell number of colony sizes indicated on the right. * denotes significant p-value as measured by Chi-Square Test. E-F. Whole blood chimerism of in-vivo transplantation assay of old (E) and young (F) LT-HSC transduced with Neo1 shRNA2 or SCR control. G. Week 20 blood lineage distribution of blood contribution from Neo1 shRNA 2 transduced young transplanted HSCs compared to SCR control.
Discussion
In this study, we selected a gene identified in our previous work as being most frequently upregulated in HSC aging and therefore a promising candidate for further investigation. The work reported here serves as a proof-of-concept in our approach to selecting candidate genes for further investigation. Not only is Neogenin expression increased in both human and mouse aged HSCs, but it is highly preferentially expressed in the most primitive LT-HSC fraction in both the old and young HSC compartment. Previous studies have shown Neogenin expression in progenitors of many tissues, including retinal axons, olfactory neuroblasts and embryonic stem cells, and its downregulation upon differentiation. In some of these studies, knock-down of Neogenin has been shown to result in a block in differentiation. Although Neogenin has been thus far regarded mainly as a migratory and neuronal protein, our experiments in HSCs and the other studies previously mentioned give strong evidence for a wider role of a marker of stem cell potential.
Repression of Neogenin affected young and aged HSCs to different degrees. Although both had an effect on proliferation in vitro, only aged HSCs showed reduced chimerism upon Neogenin engraftment. In fact, aged HSCs were unable to engraft up to 12-weeks post-transplantation. Young HSCs at 20-week post-transplantation had no difference in total blood chimerism level, but displayed a slight increase in B cell chimerism. The differences we observed in the effect of Neogenin downregulation between young and old HSCs was quite interesting and warrants further investigation. Further experiments to validate this difference and investigate molecular consequences of Neogenin downregulation are underway. It may be that the downregulation of Neogenin pushes these cells towards exhaustion. Young stem cells may withstand this longer than aged HSCs which are already at a detriment for self-renewal. The transplantation assay is notably stressful, requiring homing of cells to the bone marrow, their rapid expansion beyond homeostatic levels and simultaneous multilineage differentiation. Previous work by us and others have shown that aged HSCs perform poorly at competitive transplantation compared to young without any genetic modulation. Therefore, it may be plausible that modulation of a gene like Neogenin, which is able to regulate migration, proliferation and differentiation may affect aged HSCs to a larger degree than young. It is also possible that downregulation of Neogenin could affect a subset of HSCs dependent on a Neogenin-regulated pathway, constituting a considerably larger proportion of the aged HSC pool compared to young (due to the emergence of Neogeninhi population in aged HSCs), and that this could explain the
larger effect observed in our transplantation assay. This hypothesis is an exciting one, as a modulation that selectively affects aged HSCs with little to no consequence to young HSCs would be a promising avenue for clinical therapies. We did observe some lineage skewing in young HSCs at week 20 upon Neogenin repression, specifically an increased contribution to B-cell lineage and decrease in monocytes. Although this was not significant, the dynamics of blood repopulation still fluctuate at 20-weeks post transplantation and longer-time points are needed to reach a level of consistency.
Following the phenotypes observed, we decided to assess the functional characteristics of HSCs expressing varying levels of Neogenin. Presently, there is only one Neogenin antibody available for fluorescent activated cell sorting of viable cells (required for functional assays). In our tests on murine cells, signal from this antibody was only observed after fixation and permeabilization of murine cells. We observed no signal of this antibody when murine cells were not fixed or permeablized. We did observe signal in human Neogenin-expressing cells. The reason for this difference is not yet clear to us. Interestingly, a subset of human cord blood HSCs expressed Neogenin to a high degree (we termed these Neogeninhi cells). In vitro functional testing of these
cells revealed them to be markedly lower in both CAFC and LT-IC activity compared to Neogeninlo cells. This result was counter-intuitive, as we have seen Neogenin to
be preferentially expressed in the most primitive cells in mouse. There are some considerations to interpret this result. First, it is possible that the use of Neogenin antibody for cell sorting may inadvertently serve as a blocking antibody of the receptor by blocking residues required for binding or activation. In this scenario, we may not have measured Neogeninhi cell function but the result of blocking Neogenin activation.
It has been reported that Neogenin may function as a death/dependence receptor, wherein lack of signaling leads to apoptosis of cells, whereas Neogenin signaling maintains survival. Further work is needed to verify that this is not the case. Secondly, while the CAFC assay and LT-IC assays are well established methods to measure HSC function, their culture conditions confer a myeloid bias. If Neogenin may also affect differentiation and may also be associated with T-cells (as has been reported), it is possible that Neogenin may mark lymphoid or even T-cell biased HSCs, which less frequently differentiate into myeloid lineages measured during this assay. Other assays, such as the transplantation assay, allowing for multilineage differentiation of human HSCs may be more suitable to measure this. Lastly, we used umbilical cord blood HSCs during these assays. Although these are a rich source of functional HSCs, they are at a different developmental stage than adult HSCs. Since Neogenin is upregulated with age in both human and mouse HSCs, adult bone marrow or mobilized HSCs might contain a larger population of Neogeninhi HSCs which contain different
functional characteristics than their fetal counterparts.
Our data show that Neogenin expression is enriched in long-term HSCs and variations in its degree of expression mark HSC subsets with different stem cell potential. Knock-down of Neogenin leads to the amelioration of HSC engraftment in aged HSCs. Further investigation is needed into molecular differences between Neogeninhi
and Neogeninlo HSCs. Considering its role in development of many tissues, a reporter
or tissue specific conditional knock-out mouse may be beneficial to investigate at what stages Neogenin expression is upregulated. The possibility of isolating HSCs based on their expression of Neogenin is an exciting one which may pave the way for clinical therapy. Generation of a murine specific antibody for the external residues of Neogenin suitable for FACS would advance investigation into this approach, as the mouse has been an invaluable tool in HSC experimentation. This would allow the investigation of transplantation of Neogeninhi and lo HSCs, the consequence of their
selective depletion and the effect of Neogenin blocking antibodies on HSC function and aging.
HSC biology is well regulated and its aging phenotypes have been well documented, but are complex in nature. For example, the aged HSC pool undergoes dramatic expansion during aging but the functioning of old HSCs is reduced and they acquire the tendency to be myeloid biased. Aged HSCs have also been shown to be differentially located in the niche and have reduced homing potential. Within the umbrella that we term HSC aging there are many mechanisms and phenotypes involved. Many candidate genes investigated regulate a subset of these phenotypes, e.g. inflammation, proliferation or self-renewal. Neogenin stands out for its ability to be multifunctional and it is likely that such a multifunctional receptor may contribute in many ways to the multifaceted aging phenotypes observed. From our investigations, it is also likely that Neogenin affects HSC function not just in aged HSCs, but within LT-HSCs largely. This study therefore highlights Neogenin as a promising candidate for HSC aging and HSC biology.
Acknowledgements
This study was supported by 1) the Marie Curie ITN grant “Marriage” funded by the EU, 2) by the Mouse Clinic for Cancer and Ageing, funded by a grant from the Netherlands Organization of Scientific Research, and 3) by a Systems biology of Ageing Grant funded by Netherlands Organization for Scientific Research (NWO, grant no. 853.00.110).
The authors thank W Abdulahad, T Bijma, G. Mesander and H Moes for cell sorting assistance. Thank you to Klaas Sjollema from the UMCG Microscopy and
Imaging Center (UMIC) for assitance with confocal microscopy; E.E. Wojtowicz and J. Wudarski for valuable discussions and assistance..
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