Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow

Short Article

Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow Graphical Abstract

Authors Stefano Comazzetto, Malea M. Murphy, Stefano Berto, Elise Jeffery, Zhiyu Zhao, Sean J. Morrison

Correspondence [email protected]

In Brief Bone marrow hematopoietic stem cells reside in perivascular niches associated with sinusoids and require factors from leptin receptor+ stromal cells and endothelial cells. Morrison and colleagues show that c-kit+-restricted hematopoietic progenitors also require Stem Cell Factor made by LepR+ cells, but not endothelial cells. At least some of these restricted progenitors reside in perisinusoidal niches.

Highlights d

d

c-kit+-restricted progenitors in bone marrow require SCF from leptin receptor+ cells c-kit+-restricted progenitors in bone marrow do not require SCF from endothelial cells

d

Erythropoiesis in bone marrow requires SCF from leptin receptor+ niche cells

d

Early erythroid progenitors localize adjacent to perisinusoidal leptin receptor+ cells

Comazzetto et al., 2019, Cell Stem Cell 24, 1–10 March 7, 2019 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.stem.2018.11.022

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

Cell Stem Cell

Short Article Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow Stefano Comazzetto,1 Malea M. Murphy,1 Stefano Berto,3 Elise Jeffery,1 Zhiyu Zhao,1 and Sean J. Morrison1,2,4,* 1Children’s

Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 3Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.11.022 2Howard

SUMMARY

Hematopoietic stem cells (HSCs) are maintained in a perivascular niche in bone marrow, in which leptin receptor+ (LepR) stromal cells and endothelial cells synthesize factors required for HSC maintenance, including stem cell factor (SCF). An important question is why LepR+ cells are one hundred times more frequent than HSCs. Here, we show that SCF from LepR+ cells is also necessary to maintain many c-kit+-restricted hematopoietic progenitors. Conditional deletion of Scf from LepR+ cells depleted common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs), pre-megakaryocyte-erythrocyte progenitors (PreMegEs), and colony-forming unitserythroid (CFU-Es), as well as myeloid and erythroid blood cells. This was not caused by HSC depletion, as many other restricted progenitors were unaffected. Moreover, Scf deletion from endothelial cells depleted HSCs, but not progenitors. Early erythroid progenitors were closely associated with perisinusoidal LepR+ cells. This reveals cellular specialization within the niche: SCF from LepR+ cells is broadly required by HSCs and restricted progenitors while SCF from endothelial cells is required mainly by HSCs.

INTRODUCTION In adult mammals, hematopoiesis occurs primarily in the bone marrow, where hematopoietic stem cells (HSCs) and restricted hematopoietic progenitors are maintained throughout life. HSCs are maintained in a perivascular niche, in which leptin receptor+ (LepR+) stromal cells and endothelial cells are necessary sources of factors for HSC maintenance, including stem cell factor (SCF), Cxcl12 (Ding et al., 2012; Ding and Morrison, 2013; Greenbaum et al., 2013; Oguro et al., 2013), and pleiotro-

phin (Himburg et al., 2018). Approximately 80% of dividing and non-dividing HSCs in bone marrow are adjacent to sinusoidal blood vessels (Kiel et al., 2005; Acar et al., 2015). The niche cells we identified based on LepR expression have also been identified by others based on expression of high levels of Cxcl12 (Sugiyama et al., 2006; Omatsu et al., 2010), low levels of the Nestin-GFP transgene (Kunisaki et al., 2013), and Prx1-Cre (Greenbaum et al., 2013). Adipocytes are also a critical source of SCF for HSCs in fatty bone marrow and after myeloablation (Zhou et al., 2017). LepR+ cells arise perinatally in the bone marrow (Zhou et al., 2014). They are very rare in the first few weeks after birth but then expand in number, ultimately representing around 0.3% of young adult bone marrow cells (Ding et al., 2012; Zhou et al., 2014). In contrast, HSCs represent only around 0.003% of bone marrow cells (Harrison and Zhong, 1992). Why are LepR+ cells 100 times more numerous than HSCs? One possibility is that LepR+ cells are also a niche cell for certain restricted hematopoietic progenitors, which are far more abundant than HSCs. Like HSCs, a number of restricted progenitors express the SCF receptor, c-kit, and require SCF for their maintenance, including common myeloid progenitors (CMPs) (Akashi et al., 2000), common lymphoid progenitors (CLPs) (Kondo et al., 1997), megakaryocyte-erythrocyte progenitors (MEPs) (Akashi et al., 2000), granulocyte-macrophage progenitors (GMPs) (Akashi et al., 2000; Pronk et al., 2007), pre-megakaryocyte-erythrocyte progenitors (PreMegEs) (Pronk et al., 2007), and erythrocyte progenitors (pre-colony forming unit-erythrocytes [PreCFU-Es] and CFU-Es; Pronk et al., 2007). Together, these restricted progenitors represent around 1.5% of mouse bone marrow cells. Because 95% of the cells that express high levels of Scf in normal young adult bone marrow are LepR+ (endothelial cells express much lower levels of Scf; Zhou et al., 2014), we wondered whether c-kit+-restricted progenitors might also depend upon SCF from LepR+ cells. In contrast to the HSC niche, less is known about niches for restricted hematopoietic progenitors. A subset of early lymphoid progenitors resides near the endosteum, where they depend upon Cxcl12 synthesized by osteoblasts (Visnjic et al., 2004; Zhu et al., 2007; Ding and Morrison, 2013; Greenbaum et al., 2013). Other subsets of lymphoid progenitors depend upon perivascular niches (Tokoyoda et al., 2004; Sugiyama et al., 2006), including lymphoid progenitors that depend upon interleukin-7 Cell Stem Cell 24, 1–10, March 7, 2019 ª 2018 Elsevier Inc. 1

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

B

10

Endothelial cells

1

LepR+ cells

0.1

3

D % of cells (x10-3)

10

14

17

10

14

2

Tie2-cre; Lepr-cre;Scf Δ/FL

3

3

75 50 25

14 6

0

17 6

G

HPC-1

F

17 6

14 6

14 6

14

14

HPC-2 0.03

0.25 0.20

5

0.02

0.15 0.10

0.01

0.05

14

0

17

17

14

GMP 0.51%

MEP 0.37%

CMP 0.38%

14

0

14

17

17

14

14

0

14

Gated: Lin- c-kit+ Sca1Tie2-cre;Scf Δ/FL Lepr-cre;Scf Δ/FL

Scf Δ/FL

GMP 0.47%

MEP 0.43%

3

MPP *** **** * ***

15

14

Lepr-cre;Scf Δ/FL 3

**** **** **** ****

100

Tie2-cre;Scf Δ/FL

**

BM cellularity 125

FL/FL

Scf Δ/FL

0.25

E

Scf FL/FL

H

CD16/32

17

Scf

*** *

0.50

0

5

0

0.75

3

HSC * ******* **

15

**

1.00

% of cells

3

***

x106 cells

WBM

% of cells (x10-3)

Fold change

* 100

C

Scf 1.25

*

% of cells

Scf

1000

Fold change

A

GMP 0.46%

MEP 0.41%

CMP 0.33%

CMP 0.31%

17

Tie2-cre; Lepr-cre;Scf Δ/FL

GMP 0.35%

MEP 0.11%

17

GMP 0.32%

MEP 0.10%

CMP 0.18%

CMP 0.17%

CD34

N 50 40

8 11 11 8

9

0.1 0

8 11 11 8

9

2.0

** **

0

8 11 11 8

9

10

* 1.5

*

1.0

20

0.5

10

8 11 11 8

0.2

O Mac-1+Gr1+ in blood P B cells in blood

Mac-1+Gr1+

30

0

0.4

9

0

6

6

5

6

6

8 6 4 2 0

6

6

5

6

6

**

0.6

0.4

0.4

0.2

0.2 0

14 17 17 14 14

0

3

3

3

3

3

Q T cells in blood R Platelets in blood 4

x103 cells/μl

0

0.2

*

Myeloid colonies ** *** * **

0.6

% of cells

0.01

0.3

0.6

0.8

1200

x103 cells/μl

0.02

0.4

M

GMP

% of cells

** * *

L

MEP **** **** **** ****

0.8

% of cells

**

0.5

% of cells

0.03

K

CMP ** ** * *

x103 cells/μl

% of cells

0.04

% of cells

J

CLP

x103 cells/μl

I

3

800

2

400

1 0

6

6

5

6

6

0

8 11 11 8

9

Figure 1. SCF from LepR+ Stromal Cells Is Required to Maintain c-kit+-Restricted Progenitors in Bone Marrow (A) qRT-PCR analysis of Scf transcript levels in LepR+ stromal cells, endothelial cells, and unfractionated cells isolated from bone marrow. Data are normalized to Scf transcript levels in unfractionated bone marrow cells. (B) qRT-PCR analysis of Scf transcript levels in unfractionated bone marrow cells from mice of the indicated genotypes. The same bar colors are used for the same genotypes throughout the figure. (C) Bone marrow cellularity from two tibias and two femurs. (D–G) The frequencies of (D) HSCs, (E) MPPs, (F) HPC-1 cells, and (G) HPC-2 cells in the bone marrow (see Figure S1E and Table S3 for markers and sorting gates). (legend continued on next page)

2 Cell Stem Cell 24, 1–10, March 7, 2019

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

(IL-7) from LepR+ cells (Cordeiro Gomes et al., 2016). During regeneration after myeloablation, GMPs form clusters close to megakaryocytes and megakaryocyte depletion prolongs the persistence of GMP clusters (He´rault et al., 2017). Erythroblastic islands near macrophages are sites of terminal erythroid differentiation, and clusters near sinusoids contain more mature erythroblasts (Mohandas and Prenant, 1978; Yokoyama et al., 2003). SCF is required for the maintenance of HSCs and c-kit+restricted progenitors as well as erythropoiesis, mast cell development, and lymphopoiesis (Russell, 1979; Huang et al., 1990; Zsebo et al., 1990; Flanagan et al., 1991; Barker, 1994; Rodewald et al., 1995; Waskow et al., 2002). Experiments in culture suggest that SCF acts by promoting survival and proliferation, as well as regulating the differentiation of various hematopoietic stem and progenitor cells, including erythroid lineage cells (Toksoz et al., 1992; Muta et al., 1995; Kondo et al., 1997; Wu et al., 1997; Domen and Weissman, 2000). Here, we show that c-kit+-restricted hematopoietic progenitors, including erythroid lineage progenitors, require SCF synthesized by LepR+ cells, but not endothelial cells. This suggests that LepR+ cells are much more common than HSCs in the bone marrow because they create niches for many early restricted progenitors in addition to HSCs. RESULTS Restricted Progenitors Require SCF from LepR+ Cells By qPCR analysis, LepR+ stromal cells had 172- ± 82-fold (mean ± SD) and 35- ± 17-fold higher levels of Scf mRNA as compared to unfractionated bone marrow cells and endothelial cells, respectively (Figure 1A). To test whether LepR+ cells or endothelial cells are a necessary source of SCF for restricted progenitor maintenance in the bone marrow, we conditionally deleted Scf using Lepr-cre or Tie2-cre. Although Tie2-cre also recombines in hematopoietic cells, bone marrow hematopoietic cells do not express detectable Scf and conditional deletion of Scf from hematopoietic cells has no effect on HSC frequency or hematopoiesis (Ding et al., 2012). The presence of a single null allele of Scf in ScfD/FL mice reduced Scf transcript levels in unfractionated bone marrow cells to 52% ± 12% of the level in Scf FL/FL control mice (Figure 1B). Conditional deletion of the second Scf allele in Lepr-cre;ScfD/FL mice reduced Scf transcript levels in bone marrow cells to 12% ± 2% of Scf FL/FL control mice (Figure 1B). Conditional deletion of Scf from endothelial cells in Tie2-cre;ScfD/FL mice reduced Scf transcript levels in bone marrow cells to 44% ± 1% of Scf FL/FL control mice (Figure 1B). Conditional deletion of Scf from both endothelial cells and LepR+ cells in Tie2-cre;Lepr-cre;ScfD/FL mice reduced Scf transcript levels in bone marrow cells to 2.3% ± 0.9% of Scf FL/FL

control mice (Figure 1B). Transcripts that encode the soluble form and the membrane-bound form of SCF were both depleted in LepR+ cells from Lepr-cre;ScfD/FL mice and Tie2-cre;Leprcre;ScfD/FL mice as compared to ScfD/FL controls (Figures S1A–S1C). This is consistent with published data indicating that LepR+ cells and endothelial cells are the major sources of Scf in normal young adult bone marrow (Ding et al., 2012; Oguro et al., 2013). Relative to ScfD/FL controls, bone marrow cellularity was not significantly different in Tie2-cre;ScfD/FL mice but was significantly lower in Lepr-cre;ScfD/FL mice and in Tie2-cre;Lepr-cre; ScfD/FL mice (Figure 1C). Relative to ScfD/FL controls, the frequencies and absolute numbers of HSCs in the bone marrow were significantly reduced in Tie2-cre;ScfD/FL mice, Lepr-cre; ScfD/FL mice, and Tie2-cre;Lepr-cre;ScfD/FL mice (Figures 1D and S1G). The markers used for the isolation of each cell population are shown in Table S3, and the flow cytometric gates are shown in Figures S1D and S1E. Consistent with published studies (Ding et al., 2012; Oguro et al., 2013), competitive transplantation assays confirmed that HSC activity was significantly reduced in bone marrow from Tie2-cre;ScfD/FL mice, Lepr-cre; ScfD/FL mice, and Tie2-cre;Lepr-cre;ScfD/FL mice as compared to ScfD/FL controls (Figure S1F). The frequency and number of multipotent progenitors (MPPs) in the bone marrow were not significantly reduced in Tie2-cre;ScfD/FL mice but were significantly reduced in Lepr-cre;ScfD/FL mice and in Tie2-cre; Lepr-cre;ScfD/FL mice as compared to ScfD/FL controls (Figures 1E and S1H). This suggests that MPPs depend upon SCF synthesized by LepR+ cells, but not by endothelial cells. To begin to assess c-kit+ progenitors downstream of HSCs and MPPs, we first examined hematopoietic progenitor cells HPC-1 and HPC-2. Single-cell functional assays in vitro and in vivo suggest that these populations contain heterogeneous mixtures of restricted progenitors (Oguro et al., 2013). Relative to ScfD/FL control mice, the frequencies of HPC-1 and HPC-2 cells were not significantly altered by conditional deletion of Scf from LepR+ cells and/or endothelial cells (Figures 1F and 1G). Due to a reduction in bone marrow cellularity after deletion of Scf from LepR+ cells (Figure 1C), the absolute numbers of HPC-1 cells were significantly reduced in the bone marrow of Lepr-cre;ScfD/FL mice and Tie2-cre;Lepr-cre;ScfD/FL mice as compared to ScfD/FL controls (Figure S1I). The absolute numbers of HPC-2 cells were significantly reduced in the bone marrow of Tie2-cre;Lepr-cre;ScfD/FL mice as compared to ScfD/FL controls (Figure S1J). However, the frequencies and absolute numbers of HPC-1 and HPC-2 cells did not significantly differ between Leprcre;ScfFL/FL mice and Scf FL/FL controls (Figures S2M and S2N). The functions of HPC-1 and HPC-2 cells also did not significantly differ between Lepr-cre;ScfD/FL mice, Tie2-cre;Lepr-cre;ScfD/FL mice, and ScfD/FL controls based on the percentage of cells

(H) Representative flow cytometry plots of CMPs, MEPs, and GMPs in the bone marrow along with the percentages of bone marrow cells in each population. (I–L) The frequencies of (I) CLPs, (J) CMPs, (K) MEPs, and (L) GMPs in the bone marrow. (M) Percentage of bone marrow cells that formed myeloid colonies in culture. (N) The frequencies of Mac-1+Gr1+ myeloid cells in the bone marrow. (O–R) Numbers of (O) myeloid cells, (P) B cells, (Q) T cells, and (R) platelets per mL of blood. All data represent mean ± SD. The number of mice analyzed per genotype is shown on each bar in each panel. Statistical significance was assessed using oneway ANOVAs with Tukey’s correction for multiple comparisons (A–G and I–Q) and Kruskal-Wallis test (R; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). See also Figures S1 and S2 and Table S3.

Cell Stem Cell 24, 1–10, March 7, 2019 3

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

that formed colonies in culture, the size of the colonies, or the reconstituting activity after competitive transplantation into irradiated mice, with the exception that HPC-2 cells from mutant mice tended to form slightly larger colonies and to give higher levels of myeloid reconstitution after transplantation (Figures S1K–S1P). Overall, these results suggest that HPC-1 and HPC-2 cells exhibit a limited dependence on SCF from LepR+ cells. Deletion of Scf from LepR+ cells, but not endothelial cells, did significantly reduce the frequencies and absolute numbers of a number of other restricted progenitors in the bone marrow, including CLPs, CMPs, MEPs, and GMPs (Figures 1H–1L and S1Q–S1T). Scf deletion from LepR+ cells, but not endothelial cells, also significantly reduced the frequency of bone marrow cells that formed myeloid colonies in culture (Figure 1M), the frequency of Mac-1+Gr1+ cells in the bone marrow (Figure 1N), and the numbers of myeloid cells in the blood (Figure 1O). Deletion of Scf from LepR+ cells tended to reduce platelet counts in the blood, but the difference was not statistically significant (Figure 1R). Deletion of Scf from endothelial cells did not significantly affect the frequencies of CLPs, CMPs, MEPs, or GMPs, and there was no added effect of deleting in both LepR+ cells and endothelial cells beyond the effect of deleting only in LepR+ cells (Figures 1H–1O). Many c-kit+-restricted progenitors, therefore, require Scf from LepR+ cells, but not endothelial cells. These results did not depend on heterozygosity for the Scf null allele, as similar results were observed when we compared Leprcre;Scf FL/FL mice to Scf FL/FL controls: Lepr-cre;Scf FL/FL mice exhibited significant depletion of HSCs, CLPs, CMPs, MEPs, GMP, PreMegEs, and CFU-Es in the bone marrow based on both cell frequency and absolute number (Figures S2J–S2U). Bone marrow cells from Tie2-cre;Scf FL/FL mice exhibited significant depletion of HSCs, but not restricted progenitors (Figures S2A–S2I). These mice did not exhibit evidence of systemic SCF depletion, as coat pigmentation was normal (Figures S2V and S2W). Although CLPs were depleted by deleting Scf from LepR+ cells, downstream B lineage progenitors were not depleted when Scf was deleted from LepR+ cells and/or endothelial cells, including proB cells, preB cells, and differentiated B cells (Figures S1V–S1Z). PreB colony-forming cells were also not depleted (Figure S1U). The numbers of B and T cells in the blood were also not significantly affected by deletion of Scf from LepR+ cells and/or endothelial cells (Figures 1P and 1Q). Therefore, many restricted progenitors and differentiated cells were present in normal frequencies despite the depletion of HSCs and many ckit+-restricted progenitors in these mice. Erythropoiesis Requires Scf from LepR+ Cells The observation that CMPs and MEPs were depleted after Scf deletion from LepR+ cells (Figures 1J and 1K) raised the possibility that LepR+ cells might be a critical source of SCF for erythropoiesis in the bone marrow. Using CD150 and CD105 as markers, the CMP and MEP populations can be divided into a series of restricted erythroid progenitors, including PreMegEs, PreCFU-Es, and CFU-Es (Pronk et al., 2007; gates and markers are in Figure S3A and Table S3). Relative to ScfD/FL controls, Scf deletion from endothelial cells did not significantly affect the frequencies of any of these cell populations, but deletion from LepR+ cells significantly reduced the frequencies and absolute 4 Cell Stem Cell 24, 1–10, March 7, 2019

numbers of PreMegEs and CFU-Es in the bone marrow (Figures 2A–2E and S3B–S3D). Colony forming assays confirmed that Scf deletion from LepR+ cells, but not endothelial cells, depleted CFU-Es (Figure 2F). LepR+ cells are thus an important source of SCF for the maintenance of early erythroid progenitors. Deletion of Scf from LepR+ cells, but not endothelial cells, also depleted mature erythroid lineage cells, including proerythroblasts and basophylic erythroblasts in the bone marrow, and reduced erythroid cell counts in the blood (Figures 2G–2I, S3E, and S3F). Consistent with this, Lepr-cre;ScfD/FL mice and Tie2-cre;Lepr-cre;ScfD/FL mice (but not Tie2-cre;ScfD/FL mice) exhibited significant increases in reticulocyte frequency in the blood, as well as mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) (Figures 2J–2L), changes consistent with hypoplastic macrocytic anemia. SCF produced by LepR+ cells is, therefore, necessary for normal erythropoiesis. When bone marrow erythropoiesis does not fulfil physiological demands, erythropoiesis expands into the spleen (Paulson et al., 2011). LepR+ cells do not appear to be an important source of SCF in the spleen (Inra et al., 2015). Consistent with the reductions in bone marrow hematopoiesis in Lepr-cre;ScfD/FL mice and Tie2-cre;Lepr-cre;ScfD/FL mice, we observed significantly increased extramedullary hematopoiesis in these mice as reflected by increased splenic cellularity (Figure S3G) and increased frequencies and absolute numbers of CMPs, MEPs, GMPs, PreCFU-Es, CFU-Es, proerythroblasts, and basophylic erythroblasts in the spleen (Figures S3H–S3V). Scf Deletion from LepR+ Cells Accelerates Erythroid Progenitor Differentiation SCF can promote the survival and proliferation, and delay the differentiation, of erythroid progenitors (Muta et al., 1995; Joneja et al., 1997; Wu et al., 1997; Munugalavadla et al., 2005; McIver et al., 2016). We did not detect any effect of Scf deletion from LepR+ cells and/or endothelial cells on the frequencies of apoptotic PreMegE, PreCFU-E, or CFU-E cells based on staining for activated-caspase-3 (Figures S4A–S4C). It remains possible there was an increase in cell death we did not detect, for example, if dying cells were cleared quickly. We also detected a significant increase in plasma erythropoietin (EPO) levels after Scf deletion from LepR+ cells (Figure S4D), potentially protecting erythroid progenitors from apoptosis after Scf deletion (Waskow et al., 2004). Scf deletion from LepR+ cells and/or endothelial cells also did not affect the rate of proliferation of PreMegE (Figure 3A) or CFU-E (Figure 3C) cells based on incorporation of a 2hr pulse of 5-bromo-20 -deoxyuridine (BrdU) in vivo; however, we did observe increased BrdU incorporation by PreCFU-E cells after Scf deletion from LepR+ cells (but not endothelial cells; Figure 3B). The commitment and differentiation of erythroid progenitors are regulated by the rate of proliferation (Pop et al., 2010; Tusi et al., 2018). Because PreCFU-Es give rise to more rapidly proliferating CFU-Es in wild-type mice, the increased rate of division by PreCFU-Es after Scf deletion (Figures 3A–3C) raised the possibility that Scf deletion accelerated erythroid differentiation. To test this, we evaluated the expression of CD71 (the transferrin receptor) and c-kit in PreCFU-Es and CFU-Es. During the maturation of PreCFU-Es into CFU-Es, CD71 expression increases while c-kit expression decreases (Munugalavadla et al., 2005;

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

A

PreMegE

PreCFU-E

CFU-E

Pro Ery.

Baso Ery.

Reticulocyte

RBC

Gated: Lin- c-kit+ Sca1- CD41- CD16/32Scf Δ/FL

Scf FL/FL

B

PreCFU-E 0.034%

CFU-E 0.19%

PreCFU-E 0.036%

CFU-E 0.21%

Tie2-cre; Lepr-cre;Scf Δ/FL

Lepr-cre;Scf Δ/FL

PreCFU-E 0.044%

PreCFU-E 0.036%

CFU-E 0.054%

PreCFU-E 0.022%

CFU-E 0.036%

CD105

CFU-E 0.30%

Tie2-cre;Scf Δ/FL

PreMegE 0.10%

PreMegE 0.069%

PreMegE 0.059%

PreMegE 0.043%

PreMegE 0.030%

CD150

6

6

6

Basophylic Ery. *** **** *** ****

30

20

10

0

6

6

6

6

6

0

6

6

6

6

RBC

I 12

8

**

0

J

4

0

6

6

6

9

0.10

6

Reticulocytes **** * 8 *

0

4

80 60

4

0

4

K

6

**

4

4

MCV *** **** ******

6

6

6

6

0

0.6 0.4 0.2 0

L 20 15

40

ProEry. *** **** *** ****

0.8

4

6

6

6

6

6

MCH **** **** *** **** ***

10 5

20

6

G

% of cells

0.15

0.05

6

2

8 11 11 8

CFU-E Colonies * ** 0.20 * **

10

**** **** *****

Tie2-cre;Lepr-cre;Scf Δ/FL

0.25

0.1

6

% of cells

6

F

CFU-E ** *** **** **** ****

0.2

0.02

6

x106 cells/μl

0

% of cells

0.3

0.04

0.05

H

0.06

% of cells

% of cells

* ***

0.10

0.4

fl/cell

**

E

PreCFU-E 0.08

Lepr-cre;Scf Δ/FL

pg/cell

D

PreMegE 0.15

% of cells

C

Tie2-cre;Scf Δ/FL

% of cells

Scf Δ/FL

Scf FL/FL

8 11 11 8

9

0

8 11 11 8

9

Figure 2. Scf Deletion from LepR+ Cells Depletes Erythroid Progenitors and Causes Anemia (A) Schematic of erythroid lineage cells in the bone marrow. (B) Representative flow cytometry plots for PreMegE, PreCFU-E, and CFU-E progenitors in the bone marrow of mice of each genotype along with the percentages of bone marrow cells that fell within each population. (C–E) Frequencies of (C) PreMegE, (D) PreCFU-E, and (E) CFU-E cells in the bone marrow from mice of each genotype (see legend above these panels). (F) Frequency of CFU-E colonies formed by bone marrow cells in methylcellulose culture. (G and H) Frequencies of (G) proerythroblasts and (H) basophylic erythroblasts in bone marrow. (I–L) Red blood cell (RBC) counts (I), reticulocyte frequency (J), mean corpuscular volume (MCV) (K), and mean corpuscular hemoglobin (MCH) (L) content in the blood. All data represent mean ± SD. The number of mice analyzed per genotype is shown on each bar in each panel. Statistical significance was assessed using oneway ANOVAs with Tukey’s correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). See also Figure S3 and Table S3.

Tusi et al., 2018; compare PreCFU-E and CFU-E in Scf FL/FL control mice in Figure 3D). Scf deletion from LepR+ cells increased CD71 expression and decreased c-kit expression in PreCFU-Es and reduced c-kit expression in CFU-Es (Figures 3D–3H). This suggests that Scf deletion from LepR+ cells accelerated the differentiation of erythroid progenitors. Consistent with this possibility, SCF can delay the differentiation of erythroid progenitors in culture (Muta et al., 1995; Joneja et al., 1997; Wu et al., 1997; Munugalavadla et al., 2005; McIver et al., 2016). To further test this, we performed RNA sequencing (RNA-seq) analysis on PreCFU-E and CFU-E cells from Scf FL/FL, ScfD/FL,

and Lepr-cre;ScfD/FL mice. Only four genes significantly differed among the cells isolated from Scf FL/FL versus ScfD/FL control mice, and no Gene Ontology terms were enriched among these few differentially expressed genes. In PreCFU-Es and CFU-Es isolated from Lepr-cre;ScfD/FL mice, hundreds of genes were differentially expressed relative to cells from ScfD/FL controls. Among the gene sets that significantly increased in expression in PreCFU-E cells from Lepr-cre;ScfD/FL mice as compared to ScfD/FL mice, 4 of the top 5 categories related to erythrocyte differentiation (Table S1). Among the gene sets that significantly increased in expression in CFU-E cells from Lepr-cre;ScfD/FL Cell Stem Cell 24, 1–10, March 7, 2019 5

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

80

% BrdU+ cells

Tie2-cre;Scf Δ/FL

% BrdU+ cells

20

Scf Δ/FL

15

Lepr-cre;Scf Δ/FL

10

Tie2-cre; Lepr-cre;Scf Δ/FL

5

6

0

Scf

D

PreCFU-E

CD71+ 98%

4

G

PreCFU-E ** *

20

10 5

6

0

6

6

6

6

enrichment score

0.3 0.2 0.1 0.0

9000

10

*

3 3 3 3 3 3 0 PreCFU-E CFU-E

3000

6000

9000

Up in KO

60

40

*

4

4

3

4

3

*

0.4

NES 1.95 FDR=0.0095

0.3 0.2 0.1 0.0

20

25

3 3 3 3 3 3 PreCFU-E CFU-E

0

0

3

6

6

6

NES 1.38 FDR=0.0095

0.2 0.1 0.0

*

3 3 3 3 3 PreCFU-E CFU-E

9000

0

3000

*

9000

Down in KO

Gata2 8

* 6

*

*

200

4

*

*

Scf FL/FL Scf Δ/FL Lepr-cre; Scf Δ/FL

2

100 0

6000

Up in KO

Myb

300

*

6000

L 400

10

6

Tusi et al.-ProEry.

0.3

Down in KO 500

15

5

3000

Up in KO

20

*

6

−0.1

Sptb *

4

CFU-E

0

Down in KO

8

0

−0.1

0

RPKM

RPKM

20

20

Wong et al.-erythroblasts

NES 2.47 FDR=0.0076

0.4

Alad *

40

J

0.5

Down in KO

*

60

Tusi et al.-ProEry.

RPKM

6000

80

0

CFU-E ** * **** *

12

100

15

3

H

CFU-E

120

c-kit MFI (x103)

F

Hba-a1

*

6

CD71+ 99%

enrichment score

3

Up in KO 30

6

CD71+ 98%

0.0

K

6

Tie2-cre; Δ/FL p ; LepR-cre;Scf

CD71+ 99%

0.2

3000

LepR-cre;Scf

6

Δ/FL

CD71+ 99%

NES 2.77 FDR=0.0076

0

6

0

CD71+ 67%

PreCFU-E

0.4

6

CD71+ 65%

Wong et al.-erythroblasts 0.6

6

CD71

I enrichment score

Tie2-cre;Scf

6 Δ/FL

CD71+ 51%

20 0

6

20

CD71+ 53%

PreCFU-E * ** * *

4

6

0

40

CD71+ 44%

40

4

6

Δ/FL

60

RPKM

*

60

20

RPKM

80

6

Scf

c-kit MFI (x103)

% CD71+ cells

100

6

80

40

% CD71+ cells

c-kit

CFU-E

E

6

FL/FL

CFU-E

C

60

enrichment score

Scf

PreCFU-E * * * *

B

25

% BrdU+ cells

PreMegE

A FL/FL

3 3 3 3 3 3 PreCFU-E CFU-E

0

3 3 3 3 3 3 PreCFU-E CFU-E

(legend on next page)

6 Cell Stem Cell 24, 1–10, March 7, 2019

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

mice as compared to ScfD/FL mice, 2 of the top 5 related to erythrocyte differentiation (Table S1). We also performed gene set enrichment analysis using two curated sets of genes that are upregulated in erythroblasts as compared to earlier erythroid progenitors (Wong et al., 2011; Tusi et al., 2018). Both gene sets were significantly enriched among the genes upregulated in PreCFU-Es and CFUEs from Lepr-cre;ScfD/FL mice (Figures 3I and 3J). Genes that increase in expression during erythrocyte differentiation, including hemoglobin alpha chain 1, aminolevulinate dehydratase (Nilsson et al., 2009), and spectrin beta (Chen et al., 2009) were significantly increased in PreCFU-Es and CFU-Es from Lepr-cre;ScfD/FL mice as compared to ScfD/FL mice (Figure 3K). Conversely, genes that decrease in expression during erythrocyte differentiation, including c-Myb (Lyon and Watson, 1995) and Gata2 (Leonard et al., 1993), were significantly decreased in PreCFU-Es and CFU-Es from Lepr-cre;ScfD/FL mice (Figure 3L).

Early Erythroid Progenitors Reside in Perisinusoidal Niches with LepR+ Cells To test whether erythroid progenitors are closely associated with LepR+ cells in the bone marrow, we performed deep confocal imaging of cleared, bisected tibias (Acar et al., 2015). We localized a mixed population of early erythroid progenitors (PreCFU-E plus CFU-E cells) as Linc-kit+Sca1 CD41CD105+ cells (hereafter referred to as c-kit+CD105+ progenitors) in digitally reconstructed 3D images from Lepr-cre; Rosa26-tdTomato mice (Figures 4A and 4B). We found that 93% ± 3.7% of c-kit+CD105+ progenitors were immediately adjacent to LepR+ cells or processes from LepR+ cells, significantly more than would be expected by chance (Figure 4C). We also localized these cells in bisected bones from Cdh5creER;Rosa26-tdTomato mice to assess the localization relative to sinusoids versus arterioles (Acar et al., 2015). We found that 98% ± 0.7% of c-kit+CD105+ progenitors were most closely associated with sinusoidal blood vessels, usually within 5 mm, and the vast majority of these cells were distant from arterioles, usually more than 35 mm (Figures 4D–4F). PreCFU-E and CFU-E erythroid progenitors thus reside in perisinusoidal niches in the bone marrow, closely associated with LepR+ cells.

DISCUSSION Few studies have functionally identified the cellular sources of factors required for the maintenance of restricted hematopoietic progenitors in vivo (Ding and Morrison, 2013; Greenbaum et al., 2013; Cordeiro Gomes et al., 2016). Here, we show that many c-kit+-restricted progenitors in the bone marrow depend upon SCF synthesized by LepR+ cells. Early erythroid progenitors, and likely other c-kit+-restricted progenitors, reside in perisinusoidal niches adjacent to LepR+ cells (Figure 4). HSCs also reside mainly in perisinusoidal niches in which they depend upon SCF, Cxcl12, and pleiotrophin synthesized by LepR+ cells and endothelial cells (Ding et al., 2012; Ding and Morrison, 2013; Himburg et al., 2018). This raises the possibility that many restricted progenitors co-occupy the same niches as HSCs. It is unclear why HSCs depend upon SCF synthesized by endothelial cells (Ding et al., 2012; Ding and Morrison, 2013; Greenbaum et al., 2013; Xu et al., 2018), but most restricted progenitors do not. There are many potential differences in the presentation of SCF by endothelial cells versus LepR+ cells. The depletion of restricted progenitors after Scf deletion from LepR+ cells appears to be independent of HSC depletion. PreCFU-E cells, proB cells, preB cells, and mature B cells were not significantly depleted from the bone marrow after Scf deletion from LepR+ cells (Figures 2D, S3C, and S1V–S1Z). Therefore, not all restricted progenitors were depleted by Scf deletion, as would be expected if restricted progenitor depletion had been caused by HSC depletion. Moreover, Scf deletion from endothelial cells depleted HSCs without affecting the frequencies or absolute numbers of restricted progenitors (Figures 1D–1L and S1I–S1Z). Therefore, HSC depletion does not, in itself, lead to the depletion of restricted progenitors. Furthermore, the frequencies and functions of HPC-1 and HPC-2 progenitors that appear to be immediately downstream of MPPs, at early stages of lineage restriction, were not reduced by Scf deletion from LepR+ cells (Figures 1F, 1G, S1K–S1P, S2M, and S2N). Mice with a loss of Scf function have bone marrow hypocellularity, HSC depletion, and hypoplastic macrocytic anemia (Russell, 1979; Huang et al., 1990; Zsebo et al., 1990; Barker, 1994). Our data show that deletion of Scf from LepR+ cells recapitulated all of these phenotypes, demonstrating that

Figure 3. Scf Deletion from LepR+ Cells Accelerates Erythroid Differentiation (A–C) Incorporation of a 2-hr pulse of BrdU into (A) PreMegE, (B) PreCFU-E, and (C) CFU-E cells in the bone marrow of mice with the indicated genotypes. (D) Representative flow cytometry plots for CD71 versus c-kit expression in PreCFU-E and CFU-E cells from the bone marrow of mice with the indicated genotypes. (E) Percentages of CD71+ PreCFU-E cells from the bone marrow. (F) Mean fluorescence intensity for c-kit staining on PreCFU-E cells. (G) Percentages of CD71+ CFU-E cells. (H) Mean fluorescence intensity for c-kit staining on CFU-E cells. (I and J) Gene set enrichment analysis showing significant enrichment of genes associated with erythroblast differentiation in PreCFU-Es (I) and CFU-Es (J) from Lepr-cre;ScfD/FL mice as compared to ScfD/FL controls. FDR, false discovery rate; NES, normalized enrichment score. (K) Hemoglobin alpha chain 1, Aminolevulinate dehydratase, and Spectrin beta transcript levels in PreCFU-Es and CFU-Es from mice of the indicated genotypes (see legend on right) by RNA-seq analysis (n = 3). (L) Myb and Gata2 transcript levels in PreCFU-Es and CFU-Es by RNA-seq analysis (n = 3). Data represent mean ± SD. The numbers of mice analyzed per genotype is shown in the bars in each panel. Statistical significance was assessed using one-way ANOVAs (A–C, E, and G) or repeated-measures one-way ANOVAs (F and H) with Tukey’s correction for multiple comparisons and the DEseq2 Program with Benjamini-Hochberg corrections for multiple comparisons (K and L; *p < 0.05; **p < 0.01). See also Figure S4 and Table S1.

Cell Stem Cell 24, 1–10, March 7, 2019 7

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

A

B

C

D

E

F

Figure 4. Lineage–Sca1–c-kit+CD105+ Erythroid Progenitors Reside in Perisinusoidal Niches Adjacent to LepR+ Stromal Cells (A) Representative tiled composite confocal image of a 3-mm optical section from a bisected tibia from a Lepr-cre;Rosa26-tdTomato mouse stained with antibodies against Lineage markers and Sca1 (blue), c-kit (green), CD105 (white), and tdTomato (red). (B) Higher magnification image from (A) (white square), showing a LineageSca1c-kit+CD105+ cell (arrow) adjacent to a perisinusoidal LepR+ cell (red; * marks the sinusoid lumen). (C) Distances between LineageSca1c-kit+CD105+ cells or random spots and the closest LepR+ cells in tibias from Lepr-cre;Rosa26-tdTomato mice (n = 5). (D and E) Distances between LineageSca1c-kit+CD105+ cells or random spots and the closest sinusoids (D) or arterioles (E) in tibias from Cdh5-creER;Rosa26tdTomato mice (n = 3). (F) Percentages of LineageSca1c-kit+CD105+ cells or random spots that are closest to sinusoids versus arterioles in tibias from Cdh5-creER;Rosa26-tdTomato mice (n = 3). In (C)–(F), data represent mean ± SD. In (C)–(E), statistical significance was assessed using Kolmogorov-Smirnov tests.

LepR+ cells are a major source of SCF for erythropoiesis in the bone marrow. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

KEY RESOURCES TABLE CONTACT FOR REAGENTS AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B Primary cell culture

8 Cell Stem Cell 24, 1–10, March 7, 2019

d

METHOD DETAILS B Flow cytometry analysis and sorting of hematopoietic cells B Flow cytometric sorting of stromal cells B Bone marrow reconstitution assays B 5-bromodeoxyuridine incorporation B Analysis of apoptosis by caspase-3 staining B Measurement of EPO plasma levels by ELISA B Deep imaging of half bones B Image analysis and quantification B RNA extraction and real time qPCR B RNA-Seq library preparation and data analysis

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

d d

QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and three tables and can be found with this article online at https://doi.org/10.1016/j.stem.2018.11.022. ACKNOWLEDGMENTS S.J.M. is a Howard Hughes Medical Institute Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the Kathryn and Gene Bishop Distinguished Chair in Pediatric Research, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. S.C. was supported by an EMBO Long-Term Fellowship (ALTF 722-2015). E.J. was a postdoctoral fellow of the Damon Runyon Cancer Research Foundation (DRG-2278-16). M.M.M. was supported by a National Research Service Award from the NIH (F32 HL124947). This work was funded by the National Institute on Aging (R37 AG024945), the National Heart, Lung, and Blood Institute (HL097760), and the Cancer Prevention and Research Institute of Texas. We thank Kati Ahlqvist for help with transplantation experiments as well as Jian Xu and Andrew DeVilbiss for helpful discussions. We thank N. Loof and the Moody Foundation Flow Cytometry Facility and A. Gross for mouse colony management. AUTHOR CONTRIBUTIONS S.C., M.M.M., and E.J. performed the experiments; S.C. analyzed the data with help from Z.Z. and M.M.M. Z.Z. performed the Kolmogorov-Smirnov statistical analyses and advised on other statistical methods. S.B. analyzed the RNA-seq data. S.C. and S.J.M. conceived the experiments, interpreted the results, and wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: August 8, 2018 Revised: October 18, 2018 Accepted: November 21, 2018 Published: January 17, 2019 SUPPORTING CITATIONS The following reference appears in the Supplemental Information: Hardy et al. (1991). REFERENCES Acar, M., Kocherlakota, K.S., Murphy, M.M., Peyer, J.G., Oguro, H., Inra, C.N., Jaiyeola, C., Zhao, Z., Luby-Phelps, K., and Morrison, S.J. (2015). Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130.

DeFalco, J., Tomishima, M., Liu, H., Zhao, C., Cai, X., Marth, J.D., Enquist, L., and Friedman, J.M. (2001). Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus. Science 291, 2608–2613. Ding, L., and Morrison, S.J. (2013). Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. Nature 495, 231–235. Ding, L., Saunders, T.L., Enikolopov, G., and Morrison, S.J. (2012). Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462. Domen, J., and Weissman, I.L. (2000). Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the other. J. Exp. Med. 192, 1707–1718. Flanagan, J.G., Chan, D.C., and Leder, P. (1991). Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant. Cell 64, 1025–1035. Greenbaum, A., Hsu, Y.M., Day, R.B., Schuettpelz, L.G., Christopher, M.J., Borgerding, J.N., Nagasawa, T., and Link, D.C. (2013). CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 495, 227–230. Hardy, R.R., Carmack, C.E., Shinton, S.A., Kemp, J.D., and Hayakawa, K. (1991). Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173, 1213–1225. Harrison, D.E., and Zhong, R.K. (1992). The same exhaustible multilineage precursor produces both myeloid and lymphoid cells as early as 3-4 weeks after marrow transplantation. Proc. Natl. Acad. Sci. USA 89, 10134–10138. He´rault, A., Binnewies, M., Leong, S., Calero-Nieto, F.J., Zhang, S.Y., Kang, Y.A., Wang, X., Pietras, E.M., Chu, S.H., Barry-Holson, K., et al. (2017). Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis. Nature 544, 53–58. Himburg, H.A., Termini, C.M., Schlussel, L., Kan, J., Li, M., Zhao, L., Fang, T., Sasine, J.P., Chang, V.Y., and Chute, J.P. (2018). Distinct bone marrow sources of pleiotrophin control hematopoietic stem cell maintenance and regeneration. Cell Stem Cell 23, 370–381.e5. Huang, E., Nocka, K., Beier, D.R., Chu, T.Y., Buck, J., Lahm, H.W., Wellner, D., Leder, P., and Besmer, P. (1990). The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 63, 225–233. Inra, C.N., Zhou, B.O., Acar, M., Murphy, M.M., Richardson, J., Zhao, Z., and Morrison, S.J. (2015). A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature 527, 466–471. Joneja, B., Chen, H.C., Seshasayee, D., Wrentmore, A.L., and Wojchowski, D.M. (1997). Mechanisms of stem cell factor and erythropoietin proliferative co-signaling in FDC2-ER cells. Blood 90, 3533–3545. Kiel, M.J., Yilmaz, O.H., Iwashita, T., Yilmaz, O.H., Terhorst, C., and Morrison, S.J. (2005). SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121. Kisanuki, Y.Y., Hammer, R.E., Miyazaki, J., Williams, S.C., Richardson, J.A., and Yanagisawa, M. (2001). Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230, 230–242. Kondo, M., Weissman, I.L., and Akashi, K. (1997). Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672.

Akashi, K., Traver, D., Miyamoto, T., and Weissman, I.L. (2000). A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197.

Kunisaki, Y., Bruns, I., Scheiermann, C., Ahmed, J., Pinho, S., Zhang, D., Mizoguchi, T., Wei, Q., Lucas, D., Ito, K., et al. (2013). Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502, 637–643.

Barker, J.E. (1994). Sl/Sld hematopoietic progenitors are deficient in situ. Exp. Hematol. 22, 174–177.

Leonard, M., Brice, M., Engel, J.D., and Papayannopoulou, T. (1993). Dynamics of GATA transcription factor expression during erythroid differentiation. Blood 82, 1071–1079.

Chen, K., Liu, J., Heck, S., Chasis, J.A., An, X., and Mohandas, N. (2009). Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis. Proc. Natl. Acad. Sci. USA 106, 17413–17418. Cordeiro Gomes, A., Hara, T., Lim, V.Y., Herndler-Brandstetter, D., Nevius, E., Sugiyama, T., Tani-Ichi, S., Schlenner, S., Richie, E., Rodewald, H.R., et al. (2016). Hematopoietic stem cell niches produce lineage-instructive signals to control multipotent progenitor differentiation. Immunity 45, 1219–1231.

Lyon, J.J., and Watson, R.J. (1995). Conditional inhibition of erythroid differentiation by c-Myb/oestrogen receptor fusion proteins. Differentiation 59, 171–178. McIver, S.C., Katsumura, K.R., Davids, E., Liu, P., Kang, Y.A., Yang, D., and Bresnick, E.H. (2016). Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis. eLife 5, e17877.

Cell Stem Cell 24, 1–10, March 7, 2019 9

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

Mohandas, N., and Prenant, M. (1978). Three-dimensional model of bone marrow. Blood 51, 633–643. Munugalavadla, V., Dore, L.C., Tan, B.L., Hong, L., Vishnu, M., Weiss, M.J., and Kapur, R. (2005). Repression of c-kit and its downstream substrates by GATA-1 inhibits cell proliferation during erythroid maturation. Mol. Cell. Biol. 25, 6747–6759. Muta, K., Krantz, S.B., Bondurant, M.C., and Dai, C.H. (1995). Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation. Blood 86, 572–580. Nilsson, R., Schultz, I.J., Pierce, E.L., Soltis, K.A., Naranuntarat, A., Ward, D.M., Baughman, J.M., Paradkar, P.N., Kingsley, P.D., Culotta, V.C., et al. (2009). Discovery of genes essential for heme biosynthesis through large-scale gene expression analysis. Cell Metab. 10, 119–130. Oguro, H., Ding, L., and Morrison, S.J. (2013). SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13, 102–116. Omatsu, Y., Sugiyama, T., Kohara, H., Kondoh, G., Fujii, N., Kohno, K., and Nagasawa, T. (2010). The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche. Immunity 33, 387–399. Paulson, R.F., Shi, L., and Wu, D.C. (2011). Stress erythropoiesis: new signals and new stress progenitor cells. Curr. Opin. Hematol. 18, 139–145. Pop, R., Shearstone, J.R., Shen, Q., Liu, Y., Hallstrom, K., Koulnis, M., Gribnau, J., and Socolovsky, M. (2010). A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS Biol. 8, e1000484. Pronk, C.J., Rossi, D.J., Ma˚nsson, R., Attema, J.L., Norddahl, G.L., Chan, C.K., Sigvardsson, M., Weissman, I.L., and Bryder, D. (2007). Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell 1, 428–442. Rodewald, H.R., Kretzschmar, K., Swat, W., and Takeda, S. (1995). Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo. Immunity 3, 313–319. Russell, E.S. (1979). Hereditary anemias of the mouse: a review for geneticists. Adv. Genet. 20, 357–459. So¨rensen, I., Adams, R.H., and Gossler, A. (2009). DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113, 5680–5688. Sugiyama, T., Kohara, H., Noda, M., and Nagasawa, T. (2006). Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 25, 977–988. Tokoyoda, K., Egawa, T., Sugiyama, T., Choi, B.I., and Nagasawa, T. (2004). Cellular niches controlling B lymphocyte behavior within bone marrow during development. Immunity 20, 707–718.

10 Cell Stem Cell 24, 1–10, March 7, 2019

Toksoz, D., Zsebo, K.M., Smith, K.A., Hu, S., Brankow, D., Suggs, S.V., Martin, F.H., and Williams, D.A. (1992). Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor. Proc. Natl. Acad. Sci. USA 89, 7350–7354. Tusi, B.K., Wolock, S.L., Weinreb, C., Hwang, Y., Hidalgo, D., Zilionis, R., Waisman, A., Huh, J.R., Klein, A.M., and Socolovsky, M. (2018). Population snapshots predict early haematopoietic and erythroid hierarchies. Nature 555, 54–60. Visnjic, D., Kalajzic, Z., Rowe, D.W., Katavic, V., Lorenzo, J., and Aguila, H.L. (2004). Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 103, 3258–3264. Waskow, C., Paul, S., Haller, C., Gassmann, M., and Rodewald, H.R. (2002). Viable c-Kit(W/W) mutants reveal pivotal role for c-kit in the maintenance of lymphopoiesis. Immunity 17, 277–288. Waskow, C., Terszowski, G., Costa, C., Gassmann, M., and Rodewald, H.R. (2004). Rescue of lethal c-KitW/W mice by erythropoietin. Blood 104, 1688–1695. Wong, P., Hattangadi, S.M., Cheng, A.W., Frampton, G.M., Young, R.A., and Lodish, H.F. (2011). Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes. Blood 118, e128–e138. €ller, U., Acurio, A., Hsiao, J.G., and Lodish, H.F. (1997). Wu, H., Klingmu Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation. Proc. Natl. Acad. Sci. USA 94, 1806–1810. Xu, C., Gao, X., Wei, Q., Nakahara, F., Zimmerman, S.E., Mar, J., and Frenette, P.S. (2018). Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow. Nat. Commun. 9, 2449. Yokoyama, T., Etoh, T., Kitagawa, H., Tsukahara, S., and Kannan, Y. (2003). Migration of erythroblastic islands toward the sinusoid as erythroid maturation proceeds in rat bone marrow. J. Vet. Med. Sci. 65, 449–452. Zhou, B.O., Yue, R., Murphy, M.M., Peyer, J.G., and Morrison, S.J. (2014). Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 15, 154–168. Zhou, B.O., Yu, H., Yue, R., Zhao, Z., Rios, J.J., Naveiras, O., and Morrison, S.J. (2017). Bone marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting SCF. Nat. Cell Biol. 19, 891–903. Zhu, J., Garrett, R., Jung, Y., Zhang, Y., Kim, N., Wang, J., Joe, G.J., Hexner, E., Choi, Y., Taichman, R.S., and Emerson, S.G. (2007). Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells. Blood 109, 3706–3712. Zsebo, K.M., Williams, D.A., Geissler, E.N., Broudy, V.C., Martin, F.H., Atkins, H.L., Hsu, R.Y., Birkett, N.C., Okino, K.H., Murdock, D.C., et al. (1990). Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213–224.

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies a-mouse Gr1 Biotin

Tonbo Biosciences

clone RB6-8C5; RRID:AB_2621652

a-mouse Gr1 FITC

Tonbo Biosciences

clone RB6-8C5; RRID:AB_2621721

a-mouse Gr1 PE-efluor610

eBioscience

clone RB6-8C5; Cat. 61-5931-80

a-mouse Gr1 BV510

Biolegend

clone RB6-8C5; RRID:AB_2650931

a-mouse Gr1 PE

Tonbo Biosciences

clone RB6-8C5; RRID:AB_2621803

a-mouse CD2 Biotin

Biolegend

clone RM2-5; RRID:AB_312650

a-mouse CD2 FITC

Tonbo Biosciences

clone RM2-5; RRID:AB_2621657

a-mouse CD2 PE

Tonbo Biosciences

clone RM2-5; RRID:AB_2621728

a-mouse CD5 Biotin

Biolegend

clone 53-7.3; RRID:AB_ 312732

a-mouse CD5 FITC

Biolegend

clone 53-7.3; RRID:AB_312734

a-mouse CD5 PE

Biolegend

clone 53-7.3; RRID:AB_312736

a-mouse CD3 Biotin

Biolegend

clone 17A2; RRID:AB_2563946

a-mouse CD3 FITC

Biolegend

clone 17A2; RRID:AB_312661

a-mouse CD3 PE

Tonbo Biosciences

clone 17A2; RRID:AB_2621731

a-mouse CD3 Percp-Cy5.5

Tonbo Biosciences

clone 145-2C11; RRID:AB_2621872

a-mouse CD8a Biotin

Tonbo Biosciences

clone 53-6.7; RRID:AB_2621638

a-mouse CD8a FITC

Tonbo Biosciences

clone 53-6.7; RRID:AB_2621671

a-mouse CD8a PE

Tonbo Biosciences

clone 53-6.7; RRID:AB_2621741

a-human/mouse B220 Biotin

Tonbo Biosciences

clone RA3-6B2; RRID:AB_2621644

a-human/mouse B220 FITC

Tonbo Biosciences

clone RA3-6B2; RRID:AB_ 2621690

a-human/mouse B220 APC

Tonbo Biosciences

clone RA3-6B2; RRID:AB_ 2621574

a-human/mouse B220 Percp-Cy5.5

Tonbo Biosciences

clone RA3-6B2; RRID:AB_2621892

a-human/mouse B220 PE

Tonbo Biosciences

clone RA3-6B2; RRID:AB_2621764

a-mouse CD45.1 PE-Cy7

Tonbo Biosciences

A20; RRID:AB_2621850

a-mouse CD45.2 VioletFluor 450

Tonbo Biosciences

104; RRID:AB_2621950

a-mouse Ter119 Biotin

Tonbo Biosciences

clone TER-119; RRID:AB_2621651

a-mouse Ter119 FITC

Tonbo Biosciences

clone TER-119; RRID:AB_2621720

a-mouse Ter119 PE

Tonbo Biosciences

clone TER-119; RRID:AB_2621802

a-mouse Ter119 PE-Cy7

Biolegend

clone TER-119; RRID:AB_2281408

a-mouse Ter119 BV650

Biolegend

clone TER-119; RRID:AB_11204244

a-mouse CD71 FITC

eBioscience

clone R17217; RRID:AB_465124

a-mouse CD71 BV421

Biolegend

clone R17217; RRID:AB_10899739

a-mouse CD71 PE-Cy7

Biolegend

clone R17217; RRID:AB_2203383

a-mouse c-kit APC-eFluor780

eBioscience

clone 2B8; RRID:AB_1272177

a-mouse Sca1 Percp-Cy5.5

eBioscience

clone D7; RRID:AB_1272177

a-mouse Sca1 Alexa 488

eBioscience

clone D7; RRID:AB_493270

a-mouse CD150 PE

Biolegend

clone TC15-12F12.2; RRID:AB_313682

a-mouse CD150 PE-Cy7

Biolegend

clone TC15-12F12.2; RRID:AB_439796

a-mouse CD150 APC

Biolegend

clone TC15-12F12.2; RRID:AB_493461

a-mouse CD48 APC

Biolegend

clone HM48-1; RRID:AB_571996

a-mouse CD48 PE-Cy7

Biolegend

clone HM48-1; RRID:AB_2075049

a-mouse CD48 Alexa Fluor700

Biolegend

Clone HM48-1; RRID:AB_10612754

a-mouse CD34 FITC

eBioscience

clone RAM34; RRID:AB_465022

a-mouse IL7Ra (CD127) PE

Tonbo Biosciences

clone A7R34; RRID:AB_2621780 (Continued on next page)

Cell Stem Cell 24, 1–10.e1–e6, March 7, 2019 e1

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

a-mouse IL7Ra (CD127) PE-Cy7

Tonbo Biosciences

clone A7R34; RRID:AB_2621859

a-mouse Flt3 (CD135) PE-Cy5

Biolegend

clone A2F10; RRID:AB_2107049

a-mouse Flt3 (CD135) APC

Biolegend

clone A2F10; RRID:AB_2107050

a-mouse CD43 PE

BD Biosciences

clone S7; RRID:AB_394748

a-mouse IgM APC

eBioscience

clone Il/41; RRID:AB_469458)

a-mouse Mac-1 (CD11b) APC-efluor780

eBioscience

clone M1/70; RRID:AB_1603193

a-mouse CD16/32 (FcgRIII/II) BV510

Biolegend

clone 93; RRID:AB_2563692

a-mouse CD16/32 (FcgRIII/II) Alexa Fluor700

eBioscience

clone 93; RRID:AB_493994

a-mouse CD41 FITC

Biolegend

clone MWReg30; RRID:AB_2129746

a-mouse CD41 Alexa Fluor700

Biolegend

clone MWReg30; RRID:AB_2572129

a-mouse CD105 BV421

BD Biosciences

clone MJ/78; RRID:AB_2734710

a-mouse CD105 APC

Biolegend

clone MJ/78; RRID:AB_2687060

a-mouse CD105 Alexa 647

Biolegend

clone MJ/78; RRID:AB_2728140

a-mouse CD45 FITC

Tonbo Biosciences

clone 30-F11; RRID:AB_2621689

Goat a-mouse LepR antibody, Biotin

R&D System

BAF497; RRID:AB_2296953

a-mouse VE-cadherin (CD144) efluor660

eBioscience

clone BV13; RRID:AB_11219483

Streptavidin efluor450

eBioscience

RRID:AB_10359737

Streptavidin PE

Biolegend

RRID:AB_2571915

Rabbit anti-active caspase 3 BUV395

BD Biosciences

Cat. 564095

goat a-c-kit antibody

R&D Systems

Cat. BAF1356; RRID:AB_355961

rabbit a-dsRed/tdTomato antibody

Takara

Cat. 632496

mouse a-FITC Alexa 488 antibody

Jackson ImmunoResearch

Cat. 200-542-037; RRID:AB_2339038

F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) Dylight 405

Jackson ImmunoResearch

Cat. 711-476-152; RRID:AB_2632566

donkey a-goat IgG (H+L) Alexa 555

Thermo Fisher Scientific

Cat. A21432; RRID:AB_2535853

HI bovine serum (FBS)

Thermo Fisher Scientific

Cat. 26170043

Hank’s Balanced Salt Solution (HBSS)

Thermo Fisher Scientific

Cat. MT21022CV

Anti-APC magnetic microbeads

Miltenyi Biotech

Cat. 130-090-855

Thiazole Orange

Sigma-Aldrich

Cat. 390062

DNase I

Sigma-Aldrich

Cat. 10104159001

LiberaseDL

Sigma-Aldrich

Cat. 5466202001

5-bromo-20 -deoxyuridine

Sigma-Aldrich

Cat. B5002

NP-40

Sigma- Aldrich

Cat. I8896

Donkey serum

Jackson ImmunoResearch

Cat. 017-000-121

DMSO

Sigma-Aldrich

Cat. D4540

Trizol

Invitrogen

Cat. 15596018

Chemicals, Peptides, and Recombinant Proteins

Methocult GM M3434

Stemcell Technology

Cat. 03434

Methocult M3630

Stemcell Technology

Cat. 03630

Methocult GF M3534

Stemcell Technology

Cat. 03534

Recombinant murine thrombopoietin (TPO)

Peprotech

Cat. 315-14

Recombinant murine SCF

Peprotech

Cat. 250-03

Recombinant murine GM-CSF

Peprotech

Cat. 315-03

Recombinant murine Flt-3 ligand

Peprotech

Cat. 250-31

Tamoxifen

Sigma-Aldrich

Cat. T5648

Corn oil

Sigma-Aldrich

Cat. C8267

Benzyl alcohol

Sigma-Aldrich

Cat. 305197

Benzyl benzoate

Sigma-Aldrich

Cat. B6630 (Continued on next page)

e2 Cell Stem Cell 24, 1–10.e1–e6, March 7, 2019

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

BD APC BrdU Flow Kit

BD Biosciences

Cat. 552598

BD Fixation/Permeabilization Solution Kit

BD Biosciences

Cat. 554714

Mouse Erythropoietin Quantikine ELISA Kit

R&D Systems

Cat. MEP00B

Critical Commercial Assays

High-Capacity cDNA Reverse Transcription kit

Applied Biosystem

Cat. 4368814

RNeasy Micro Kit

QIAGEN

Cat. 74004

SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Takara

Cat. 634413

this paper

GEO: GSE122468

Deposited Data RNaseq analysis Experimental Models: Organisms/Strains Scf FL (Kitltm2.1Sjm/J, RRID:IMSR_JAX:017861)

Ding et al., 2012

N/A

Scf GFP (Kitltm1.1Sjm/J, RRID:IMSR_JAX:017860)

Ding et al., 2012

N/A

ScfD (Kitltm2.2Sjm/J, RRID:MGI:5306348)

Ding et al., 2012

N/A

Tie2-Cre (B6.Cg-Tg(Tek-cre)1Ywa/J, RRID:IMSR_JAX:008863

Kisanuki et al., 2001

N/A

LepR-Cre (B6.129(Cg)-Leprtm2(cre)Rck/J, RRID:IMSR_JAX:008320)

DeFalco et al., 2001

N/A

Rs26-tdTomato (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato) Hze/J, RRID:IMSR_JAX:007909)

Jackson Laboratory

JAX:007909

Cdh5-CreER (B6.CBA-Tg(Cdh5-cre/ERT2)1Rha, RRID: IMSR_TAC:13073)

So¨rensen et al., 2009

N/A

IDT

Table S2

FlowJo v.10.4

Flowjo, LLC

N/A

Imaris Bitplane v.8.2

Bitplane

N/A

Graphpad Prism v.6

Graphpad

N/A

Oligonucleotides qRT-PCR and genotyping primers Software and Algorithms

CONTACT FOR REAGENTS AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sean Morrison ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice Scf GFP (Kitltm1.1Sjm/J, RRID:IMSR_JAX:017860), ScfFL (Kitltm2.1Sjm/J, RRID:IMSR_JAX:017861), ScfD (Kitltm2.2Sjm/J, RRID:MGI: 5306348) were generated in our laboratory (Ding et al., 2012), Tie2-cre (B6.Cg-Tg(Tek-cre)1Ywa/J, RRID:IMSR_JAX:008863) (Kisanuki et al., 2001) mice, Lepr-cre (B6.129(Cg)-Leprtm2(cre)Rck/J, RRID:IMSR_JAX:008320) (DeFalco et al., 2001) mice, and Rosa26tdTomato (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, RRID:IMSR_JAX:007909) mice were obtained from Jackson Laboratory. Cdh5-creER (B6.CBA-Tg(Cdh5-cre/ERT2)1Rha, RRID:IMSR_TAC:13073) transgenic mice were provided by Dr. Ralf H. Adams (So¨rensen et al., 2009). Mice were all backcrossed at least six times onto a C57Bl/Ka background and were maintained on this background. Germline Scf loss-of-function was achieved using the Scf GFP or the ScfD allele. We used 8-16 week-old age-matched mice in all experiments. To induce Cre recombinase activity, Cdh5-creER;Rosa26-tdTomato mice received an intraperitoneal injection of 2mg of tamoxifen dissolved in corn oil every second day for 10 days (5 injections total). All mice were housed in AAALAC-accredited, specific-pathogen-free animal care facilities at the University of Texas Southwestern Medical Center (UTSW). All procedures were approved by the UTSW Institutional Animal Care and Use Committee. Primary cell culture To assess the frequency of colony-forming units erythroid (CFU-E), 1x105 total bone marrow cells or 2x105 total spleen cells were plated in Methocult GM M3434 medium supplemented with 10ng/ml of TPO and 1X penicillin/streptomycin in 6-well plates. To assess the frequency of colony-forming units granulocyte/macrophage (CFU-GM), 1x104 total bone marrow cells were plated in Methocult GF M3534 medium supplemented with 10ng/ml Flt-3 ligand, 25ng/ml of GM-CSF, and 1x penicillin/streptomycin in 6-well plates. To assess the frequency of CFU-preB, 1x105 total bone marrow cells were plated in Methocult M3630 medium supplemented Cell Stem Cell 24, 1–10.e1–e6, March 7, 2019 e3

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

with 20ng/ml of Flt-3 ligand, 100ng/ml SCF, and 1X penicillin/streptomycin in 6-well plates. CFU-E colonies were counted after 3 days using an inverted microscope. CFU-GM and CFU-preB colonies were counted after 8 days using an inverted microscope. To assess the colony-forming capacity of HPC-1 cells and HPC-2 cells, 200 cells were double sorted by flow cytometry and plated in Methocult GM M3434 medium supplemented with 10ng/ml of TPO and 1X penicillin/streptomycin in 6-well plates. Colonies were counted after 8 days using an inverted microscope. METHOD DETAILS Flow cytometry analysis and sorting of hematopoietic cells Bone marrow was flushed from two tibias and two femurs using staining medium (HBSS supplemented with 2% bovine serum) and dissociated into a single cell suspension by gently triturating with a 23 gauge needle. Spleens were mechanically dissociated by crushing through a 40 mm cell strainer using staining medium. Cells were counted, and then stained with antibodies at 4 C for 30 minutes (except for anti-CD34 that was incubated for 90 minutes). For staining of HSCs and restricted hematopoietic progenitors, cells were first stained with antibodies against lineage markers (biotin-conjugated antibodies against CD2, CD3, CD5, CD8a, B220, Ter119, and Gr1), washed with staining medium and then resuspended in fluorophore-conjugated antibodies against c-kit, Sca1, CD150, CD48, CD34, CD16/32, Flt3, IL7Ra, and efluor450-conjugated streptavidin. For erythroid progenitors, cells were stained with fluorophore-conjugated antibodies against lineage markers (CD2, CD3, CD5, CD8a, B220, Ter119, and Gr1), c-kit, Sca1, CD150, CD41, CD16/32, CD71, and CD105. For analysis of lymphoid and myeloid cells, cells were stained with fluorophore-conjugated antibodies against Mac-1, Gr1, B220, CD43, and IgM. For erythroblasts, cells were stained with fluorophore-conjugated antibodies against CD71 and Ter119. Cells were then analyzed using a FACSAria II SORP (BD Biosciences) or a FACSAria Fusion SORP (BD Biosciences) cytometer. To sort erythroid progenitors, HPC-1 cells and HPC-2 cells, tibias, femurs, pelvises, and spines were collected and crushed using a mortar and pestle. Cells were resuspended in staining medium and filtered through a 40 mm cell strainer. The cells were stained with APC-efluo780 conjugated anti-c-kit antibody and c-kit+ cells were enriched using anti-APC paramagnetic microbeads (Miltenyi Biotec). For erythroid progenitors, cells were then stained with fluorophore-conjugated antibodies against lineage markers (CD2, CD3, CD5, CD8a, B220, Ter119, and Gr1), Sca1, CD150, CD41, CD16/32, CD105. For HPC-1 and HPC-2, cells were stained with fluorophore-conjugated antibodies against lineage markers (CD2, CD3, CD5, CD8a, B220, Ter119, and Gr1), Sca1, CD150, and CD48. Cells were isolated by two successive rounds of sorting to ensure purity using a FACSAria II cytometer. For peripheral blood analysis, 50 mL of blood were collected from the tail vein or from the heart before euthanasia and mixed with 5mL of 0.5M EDTA to prevent clotting. Platelets, RBC, MCV and MCH were evaluated using a Hemavet HV950 (Drew Scientific). To determine reticulocyte frequency in the blood, 2mL of blood were stained in 1ml of 0.1mg/ml Thiazole Orange in HBSS containing 2mM EDTA and 0.02% sodium azide. Samples were incubated for 1h at room temperature, and then analyzed by flow cytometry. For myeloid and lymphoid cells analysis, red blood cells were lysed and samples were washed in 5ml of staining medium. Cells were then stained with fluorophore-conjugated antibodies against B220, CD3, Mac-1 and Gr1 and analyzed using a FACS Canto cytometer. Dead cells were identified and gated out of all analyses by staining with DAPI or with propidium iodide after antibody staining. Flow cytometric sorting of stromal cells Bone marrow stromal cells were enzymatically digested from flushed tibias and femurs as previously described (Zhou et al., 2014). Briefly, tibias and femurs were gently flushed and then digested in HBSS plus calcium and magnesium supplemented with DNase I (200U/ml) and LiberaseDL (250mg/ml), agitating for 30 minutes at 37 C. Bone marrow fragments were allowed to sediment for 1min, then the cell suspension was collected. Cell were washed once in staining medium and incubated with a biotin-conjugated goat antiLepR antibody for 90 min at 4 C. Cells were washed and then incubated with fluorophore-conjugated antibodies against CD45 and Ter119 and PE-conjugated streptavidin. Endothelial cells were stained by intravenous injection of 10mg of anti-VE-cadherin antibody 5-10 minutes before killing the mice. Cells were purified with two successive rounds of sorting to ensure high purity using a FACSAria II SORP cytometer. Bone marrow reconstitution assays Recipient (CD45.1/CD45.2) mice were irradiated using an XRAD 320 X-ray irradiator (Precision X-Ray Inc.) with two doses of 540 rad at least 4h apart. For whole bone marrow transplantation, 500,000 unfractionated bone marrow cells from donor (CD45.2) and competitor (CD45.1) mice were mixed and injected intravenously through the retro-orbital venous sinus. Every 4 weeks until at least 16 weeks after transplantation, blood was collected from the tail vein and subjected to ammonium-chloride potassium chloride red cell lysis. Cells were then stained with antibodies against CD45.1, CD45.2, Mac-1, Gr1, B220, and CD3 to evaluate levels of donor cell engraftment in the myeloid, B, and T cell lineages. For transplantation of HPC-1 cells and HPC-2 cells, 200 HPC-1 or HPC-2 cells from donor (CD45.2) mice were mixed with 200,000 bone marrow cells from competitor (CD45.1) mice and injected intravenously through the retro-orbital sinus into CD45.1/CD45.2 recipients. Levels of donor cell engraftment were evaluated in the bone marrow of recipient mice at 14 days after transplantation by staining with antibodies against CD45.1, CD45.2, Mac-1, Gr1, B220, and CD3.

e4 Cell Stem Cell 24, 1–10.e1–e6, March 7, 2019

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

5-bromodeoxyuridine incorporation Mice were intraperitoneally injected with 0.1mg/g body mass of 5-bromo-20 -deoxyuridine (BrdU) and killed 2 hours after injection. Bone marrow cells were obtained and c-kit+ cells were enrichment as described above, Lineage-c-kit+Sca1- cells were sorted in purity mode into a 1.5ml eppendorf tube using a FACSAria II flow cytometer. Staining for BrdU incorporation was performed using the BD APC BrdU Flow Kit following the manufacturer’s instructions. BrdU levels in each sorted cell population were analyzed using a FACSAria II flow cytometer. Analysis of apoptosis by caspase-3 staining After isolation and counting of cells from the bone marrow, 5x106 total bone marrow cells were stained with fluorophore-conjugated antibodies against lineage markers (CD2, CD3, CD5, CD8a, B220, Ter119, and Gr1), Sca1, CD150, CD41, CD16/32 and CD105. Cells were then washed and fixed using the BD Fixation/Permeabilization Solution Kit according to the manufacturer’s instruction. Cells were then stained using a rabbit anti-activated caspase-3 antibody. Data were acquired using a FACSAria II flow cytometer. Measurement of EPO plasma levels by ELISA Plasma was obtained from peripheral blood after centrifugation. EPO levels were measured using the Mouse Erythropoietin Quantikine ELISA Kit following the manufacturer’s instructions. Deep imaging of half bones Sample preparation and immunostaining of bisected (half) tibias were performed as previously described (Acar et al., 2015). Briefly, tibias from Lepr-cre;Rosa26-tdTomato or Cdh5-creER;Rosa26-tdTomato mice were fixed in 4% paraformaldehyde solution in PBS for 6 hours with gentle rocking at 4 C. After fixation, bones were embedded into OCT embedding medium. Each tibia was cut in two using a cryostat and then washed three times in PBS. Half bones were blocked using whole mount staining medium (PBS with 5% donkey serum, 0.5% NP-40, and 10% DMSO) overnight at room temperature. Half bones were then stained in whole mount staining medium with antibodies against lineage markers (FITC-conjugated CD3, B220, Ter119, Gr1, CD41), as well as goat anti-c-kit and rabbit anti-DsRed/Tomato antibodies for three days at room temperature. The samples were washed three times for 5 minutes in PBS and then overnight in PBS. Half bones were then stained with anti-Sca1 Alexa 488, anti-CD105 Alexa-647, mouse anti-FITC Alexa 488, donkey anti-goat Alexa-555 and donkey anti-rabbit DyLight-405 antibodies for three days at room temperature. The samples were washed three times for 5 minutes in PBS and then overnight in PBS. Bone clearing was performed in eppendorf tubes, gently rotating at room temperature. Samples were dehydrated through a methanol series before clearing with Murray’s clear (1:2 Benzyl Alcohol: Benzyl Benzoate; BABB) solution. Half bones were mounted in BABB and three dimensional images of the bone marrow were acquired using a Confocal/Multiphoton Zeiss LSM880 Inverted microscope with a Zeiss LD LCI Plan-Apo 253/0.8 multiimmersion objective lens. Image analysis and quantification Confocal half bone images were rendered in three dimensions and analyzed using Bitplane Imaris V8.2. Automated identification of Lin-c-kit+Sca1-CD105+ erythroid progenitors required a workflow including several steps. First, we subtracted the Lineage marker signal from the CD105 signal using the channel arithmetic function in Imaris to identify Lin-CD105+ cells. Second, we inverted the c-kit channel, setting the signal for positive cells above a predetermined threshold to zero, and then subtracted the inverted c-kit signal from the Lin-CD105+ channel using the channel arithmetic function. This allowed us to identify Lin-c-kit+Sca1-CD105+ erythroid progenitors in a single channel. Finally, we created a digital surface for Lin-c-kit+Sca1-CD105+ progenitors using the Imaris surface function, by manually selecting image intensity values that would distinguish Lin-c-kit+Sca1-CD105+ progenitors from other cells and from debris or staining artifacts. The original, unprocessed images were then manually checked to verify that each cell identified as a Lin-c-kit+Sca1-CD105+ progenitors had the appropriate staining patterns and morphology in each channel. To determine the distances of Lin-c-kit+Sca1-CD105+ cells to tdTomato+ cells (from Lepr-cre;Rosa26-tdTomato and Cdh5-creER;Rosa26-tdTomato mice), we used the Imaris Surface function to create a digital surface for LepR+ cells or endothelial cells based on Tomato signal intensity. To distinguish between arterioles and sinusoids in Cdh5-creER;Rosa26-tdTomato bone marrow samples, the tdTomato+ channel was first segmented manually based on blood vessel morphology and then masked to produce two channels, one representing the tdTomato+ signal from arterioles and the other representing the tdTomato+ signal from sinusoids. Three-dimensional distances between Lin-c-kit+Sca1-CD105+ cells and these digital surfaces were calculated using the Imaris Distance Transform MATLAB XTension. To generate random spot distributions in three dimensional space, we used the random number generator function of Microsoft Excel to create random combinations of 3 numbers representing X, Y, and Z spatial coordinates. These coordinates were then imported as 8mm spots using the Imaris Create Spots From File Python XTension. To ensure that random spots were only distributed over hematopoietic regions of the bone marrow we excluded regions of bone, blood vessel lumens, and poorly stained areas from the analysis. RNA extraction and real time qPCR For total bone marrow extracts, bone marrow was isolated from tibias by centrifugation at 3000 RPM for 3 minutes at 4 C. The cell pellet was then resuspended in Trizol and stored at 80 C. Total RNA was isolated following the manufacturer’s instructions. 1 mg of RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit. For sorted stromal and endothelial cells, Cell Stem Cell 24, 1–10.e1–e6, March 7, 2019 e5

Please cite this article in press as: Comazzetto et al., Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow, Cell Stem Cell (2018), https://doi.org/10.1016/j.stem.2018.11.022

5,000-10,000 cells were sorted in staining medium, spun down, and resuspended in Trizol. Total RNA was extracted and reverse transcribed as above. Primers used for qPCR are listed in Table S2. Transcript levels were normalized to Actin (Actb) and fold change was calculated using the DCt method. RNA-Seq library preparation and data analysis 5,000-10,000 erythroid progenitors were sorted in tubes containing RLT buffer from the RNeasy Micro Kit and stored at 80 C. Total RNA was extracted following the kit instructions, and RNA integrity and concentration were measure using a Bioanalyzer. Two to six ng of RNA were used to generate a cDNA library using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Samples were sequenced with a NextSeq 500 (Illumina) using a 75bp single-read setup. Adaptor removal and quality trimming were performed using Trimmomatic. Reads were aligned to the mouse mm10 reference genome using STAR 2.5.2b. Gencode vM18 annotation was used as reference to build STAR indexes and alignment annotation. For each sample, a BAM file including mapped and unmapped reads with spanning splice junctions was produced. Secondary alignment and multi-mapped reads were further removed using in-house scripts. Only uniquely mapped reads were retained for further analyses. Quality control metrics were performed using RseqQC using the mm10 gene model provided . These steps include: number of reads after multiple-step filtering, ribosomal RNA reads depletion, and defining reads mapped to exons, UTRs, and intronic regions. Picard tool was implemented to refine the QC metrics (http://broadinstitute.github.io/picard/). Gene level expression was calculated using HTseq version 0.9.1 using intersection-strict mode by gene. Counts were calculated based on protein-coding gene annotation from the Gencode vM18 annotation file. RPKM (reads per kilobase of transcript per million reads mapped) values were calculated using edgeR. Length was curated using the protein-coding gene annotation from the Gencode vM18 annotation file. RPKM values were filtered for downstream differential and co-expression analyses using a ‘‘by condition’’ RPKM cutoff. Briefly, a gene is considered expressed if the RPKM > 0 in all three biological replicates (e.g., in ScfD/FL) in any of the cell types analyzed. Differential expression between control and mutant samples was assessed using the DESeq2 package in R. All differentially expressed genes with an FDR % 0.05 and log2(fold change) R j0.3j (1.23 fold change) were retained. A permutation test was also applied using 1000 permuted experiments. None of these permuted analyses showed the same genes differentially expressed (permutation p < 0.001). The functional enrichment of GO terms among differentially expressed genes was assessed using ToppGene. A Benjamini-Hochberg FDR (p < 0.05) was applied for a multiple comparisons adjustment. Gene set enrichment analysis (GSEA) and visualization were performed using the fgsea package in R (https:// github.com/ctlab/fgsea) using two gene signatures for genes highly expressed in erythroblasts (Wong et al., 2011) and proerythroblasts (Tusi et al., 2018). QUANTIFICATION AND STATISTICAL ANALYSIS Data in all figures were obtained in at least three independent experiments using different mice, as indicated in each figure panel. Data are shown as mean ± standard deviation. Flow cytometry data were analyzed using Flowjo v.10.4 (Flowjo, LLC). Prior to analyzing the statistical significance of differences among treatments, we tested for normality in the data distributions using the D’Agostino-Pearson omnibus test for samples with n > 8 or the Shapiro-Wilk normality test for smaller samples. We also tested for similar variability among treatments using an F-test (when comparing two samples) or Levene’s median tests (when comparing more than two samples). When data were normally distributed and variability was similar among treatments, we used parametric tests to assess statistical significance. When the data significantly deviated from normality (p < 0.01) or variability was significantly different (p < 0.01) among treatments, we log2 transformed the data and tested for normality and variability. If the log2 transformed data did not significantly deviate from normality and variability among treatments did not significantly differ, we performed parametric tests to assess the statistical significance of differences among treatments using the log2 transformed data. If the log2 transformed data did significantly deviate from normality or similar variability among treatments, we performed non-parametric tests on the non-transformed data. Statistical analyses were performed using one-way ANOVAs with Tukey’s corrections for multiple comparisons and Kruskal-Wallis non-parametric tests when comparing more than two treatments (Graphpad Prism v.6). We used unpaired Student’s t tests with Welch’s correction when comparing two treatments (Graphpad Prism v.6). For transplantation experiments in Figure S1F, we used a non-parametric mixed model, nparLD, to test for differences in donor cell reconstitution levels among cells of different genotypes. In Figures 4C–4E, we applied a normalized two-sample Kolmogorov-Smirnov test as follows. Distances to erythroid progenitors or random spots within each biological replicate were pooled to generate 100 bins of distances, each bin corresponding to 1% of the maximum distance for each biological replicate. The frequency of erythroid progenitors or random spots in each bin was calculated for each biological replicate, then used to perform a two-sample Kolmogorov-Smirnov test using MATLAB. DATA AND SOFTWARE AVAILABILITY The accession number for the RNA-seq data generated in this paper from sorted PreCFU-E and CFU-E cells from Scf FL/FL, ScfD/FL and Lepr-cre;ScfD/FL are accessible at GEO: GSE122468. Software used to analyze the data is described in the Method Details section.

e6 Cell Stem Cell 24, 1–10.e1–e6, March 7, 2019