Probing hematopoietic stem cell function using serial transplantation: Seeding characteristics and the impact of stem cell purification

Accepted Manuscript Probing hematopoietic stem cell function using serial transplantation; seeding characteristics and the impact of stem cell purification Alexandra Rundberg Nilsson, Cornelis JH. Pronk, David Bryder PII:

S0301-472X(15)00169-1

DOI:

10.1016/j.exphem.2015.05.003

Reference:

EXPHEM 3260

To appear in:

Experimental Hematology

Received Date: 26 March 2015 Revised Date:

7 May 2015

Accepted Date: 14 May 2015

Please cite this article as: Rundberg Nilsson A, Pronk CJ, Bryder D, Probing hematopoietic stem cell function using serial transplantation; seeding characteristics and the impact of stem cell purification, Experimental Hematology (2015), doi: 10.1016/j.exphem.2015.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT •

HSCs distribute unevenly in individual bones following transplantation.

•

Transplantation of wBM compared to purified HSCs causes a Tlymphoid bias.

•

HSC purification is advantageous when conducting serial

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transplantation.

ACCEPTED MANUSCRIPT

Probing hematopoietic stem cell function using serial transplantation; seeding characteristics and the

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impact of stem cell purification

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Alexandra Rundberg Nilsson1,3, Cornelis JH Pronk1,2,3 and David Bryder1,3

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Lund University, Medical Faculty, Institution for Laboratory Medicine, Division of Molecular

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Hematology, Sölvegatan 19, BMC B12, 221 84 Lund, Sweden

Department of Pediatric Oncology/Hematology, Skåne University Hospital, 221 85 Lund,

Sweden

Lund Stem Cell Center, Biomedical Center B10, Klinikgatan 26, 221 84 Lund, Sweden

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Corresponding author: Alexandra Rundberg Nilsson, Lund University, Division of Molecular Hematology, BMC B12, Klinikgatan 26, 221 84 Lund, Sweden,

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+46 46 222 03 13, [email protected]

Word count: 1,609

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ACCEPTED MANUSCRIPT INTRODUCTION

Bone marrow-residing hematopoietic stem cells (HSCs), though low in frequency, hold the capacity to generate effector cells of all blood cell lineages

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in a highly proliferative and dynamic manner throughout life [1, 2]. Apart from their more direct therapeutic potential, including serving as vehicles for gene therapy, expansion for transplantation purposes and dissections of their roles

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in disease development, HSCs have also served as a model system to study multiple aspects of somatic stem cell behavior [3]; a foundation established

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using murine in vivo transplantation assays more than half a century ago [4]. Competitive [2] and serial [5] transplantation experiments using whole bone marrow (wBM) cells have given insights to the performance of normal and genetically manipulated HSCs, while more recent approaches to isolate HSCs

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to high purity using flow cytometry [6-9] allow for more direct evaluation of HSC activity. Still, functional evaluation of HSCs via assessment of their multilineage reconstitution capacity following HSC transplantation remains a

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cornerstone in the experimental work-up, with serial transplantation

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experiments considered to most faithfully reflect long-term HSC function [10, 11]. Despite its extensive use, we noted that serial transplantations are not conducted uniformly in between studies, in that either i) primary recipientderived sorted HSCs or ii) wBM are used for transplantation to secondary recipients, in addition to differences with regard to the types (i.e. different phenotypes) of cells transferred. This could potentially impede correct evaluation of the studied variable, as was previously exemplified in a setting of JunB deficiency, where transplantation of wBM or purified HSCs led to

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ACCEPTED MANUSCRIPT fundamentally distinct conclusions with regards to HSC performance [12][13]. As such dramatic differences can be caused solely by a single experimental variable, we decided to dissect the impact of the transplantation approach

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itself on the quantitative and qualitative performance of HSCs.

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ACCEPTED MANUSCRIPT RESULTS AND DISCUSSION

HSCs are distributed unevenly following transplantation Serial transplantation of wBM is routinely conducted with BM isolated

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from a limited number of bones. This strategy assumes similar chimerism levels in the bones of individual mice, which is not obvious. To begin to detail the HSC distribution following transplantation, we examined individual bones

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of competitively transplanted recipients. This revealed major variances in phenotypic HSC chimerism level (HSC gating strategy, Figure 1a) between

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individual bones of the same recipient mice (Table 1, Supplementary table 1), suggesting that investigations of a low proportion of BM might lead to results that are not representative for the overall HSC chimerism level of the investigated mouse. To directly assess the observed variations in HSC

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chimerism between individual bones, we conducted serial transplantation of wBM from separate bones (tibia-, femur- and hipbones) from three individual mice. Peripheral blood (PB) granulocyte chimerism levels in secondary

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recipients receiving wBM from individual bones generally mimicked the HSC

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chimerism levels of the donor bone (Table 1), confirming a correlation between the phenotypic HSC frequencies observed between individual bones and HSC function. Previous studies have indicated that some degree of HSC migration occurs during steady state [14, 15], although probably at low rates [16]. In addition, it has been reported that the distribution of different HSC clones

persists

asymmetrically

following

transplantation

[17],

further

suggesting that HSC recirculation is limited also in a transplantation scenario.

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ACCEPTED MANUSCRIPT In agreement with this, our analysis indicated that any potential recirculation of HSCs does not cause uniformity of donor HSC distribution (Table 1). In this context it is important to stress that BM cells isolated from all tibia-, femur-, and hipbones still only represents about 20 percent of the total

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BM of a mouse [18]. Transplantation of equal numbers of sorted HSCs would at least compensate for the confounding factor of uneven HSC frequencies across bones, though perhaps not fully for a possible uneven distribution of

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HSC clones that differ qualitatively. As a consequence, regardless of using BM for analysis or transplantation, isolation of cells from multiple bones would

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be preferable to better approach the average reconstitution in the primary host.

Serial transplantation of whole bone marrow as opposed to purified

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HSCs complicates evaluations of long-term stem cell function One argument for using wBM in serial transplantation experiments to assess HSC function is that this approach does not risk excluding

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phenotypically undefined HSCs [19]. However, co-transplantation of long-lived

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mature and/or progenitor blood cells [20-23] might skew lineage readouts in secondary recipients receiving wBM, which could confound interpretations on HSC behavior/output. Therefore, we next evaluated the lineage distributions of donor-derived cells (CD11b+ myeloid cells, CD19+ B-cells, and CD3+ Tcells) in primary (Figure 1b) and serially (Figure 1c) transplanted mice receiving wBM or sorted HSCs. In both scenarios, we observed a relative Tlymphoid bias at the expense of myeloid cells in recipients receiving wBM compared to sorted HSCs, which supported the interpretation that not all

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ACCEPTED MANUSCRIPT donor-derived cells in recipients of wBM descend from the serially transplanted HSCs. This interpretation is further supported by the recent observations that progenitor subsets can actively contribute to hematopoiesis long-term [24, 25].

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Another potential drawback with the use of wBM for transplantation, and from such strategies attempting to deduce effects at the level of HSCs, is the risk of assuming functional differences based on different levels of

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chimerism. Such differences could obviously be achieved by different frequencies of HSCs transplanted. For instance, wBM transplantation

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experiments of aged BM cells could argue for an unaltered, or even increased HSC repopulation activity [26]. However, this has been explained by an expansion of the frequencies of HSCs in aged BM [27] and a progressively declining HSC function on a per cell basis with age [28]. Therefore, using the

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same numbers of sorted HSCs is preferable to limit the influence of frequency variances, while at the same time permitting for more direct evaluations of HSC function. Furthermore, we would like to highlight the importance of

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keeping biological replicates separate and not pool BM of the primary

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recipient material to be used for serial transplantation, as spontaneous acquisition of a particular feature, for instance a proliferative disorder in one recipient, would be transferred to all recipients of that group.

Approaches to enhance HSC-derived chimerism levels in secondary recipients One drawback with serial transplantation of HSCs is the potential risk of low chimerism levels in secondary recipients, often caused by the

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ACCEPTED MANUSCRIPT competitive advantage of the freshly isolated competition over the serially transplanted HSCs [29, 30]. Such low levels of reconstitution can potentially limit evaluation of HSC function and/or obscure possible biological effects. We envisioned two approaches to address this issue. Following non-competitive

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primary transplantation of wBM, a maximal number of HSCs can be recovered from primary recipients and co-infused with fresh competitor cells, thereby ensuring adequate chimerism levels in secondary recipients (Figure 2).

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Additionally, this approach would maximize self-renewal of the test HSCs in the primary hosts, as competitor HSCs would not be present in the primary

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recipients. However, if biological or technical reasons hamper isolation of sufficient numbers of sorted HSCs for secondary transplantation, an alternative approach could be to use previously transplanted bone marrow as competitor cells (Figure 1d). Either of these approaches increases the

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contribution of the serially transplanted HSCs.

In summary, we have shown that HSCs distribute unevenly between separate bones of hosts following transplantation, with the consequence that

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serial transplantation of wBM can result in the transfer of host chimerism that

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is not representative to that observed in primary hosts. Furthermore, serial transplantation of wBM, when compared to purified HSCs, was associated with a relative lymphoid bias among donor-derived cells. This suggests that co-transplantation of long-lived mature blood cells and/or progenitor cells contribute to the production of mature cells long-term post transplantation, with implications for evaluating the differentiation potential of transplanted HSCs. We demonstrate that serial transplantation of sorted HSCs (Figure 2) overcomes these issues and enables a more direct evaluation of long-term

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ACCEPTED MANUSCRIPT HSC function on a per cell basis, as this eliminates confounding factors such as

HSC

frequency

differences

in

primary

hosts

and/or

short-term

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contributions.

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ACCEPTED MANUSCRIPT MATERIAL AND METHODS

Mice and isolation of bone marrow 8-12 week old CD45.1+ B6SJL, CD45.2+ C57CL/6, or CD45.1+CD45.2+

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B6SJL/C57CL/6 mice were used as recipients and donors throughout the experiments. BM was isolated from tibia-, femur- and hipbones as described previously [31]. Mice were maintained at the animal facilities of Biomedical

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Center at Lund University, and all experiments were performed with consent

Bone marrow transplantation

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from a local ethics committee.

HSCs were isolated as previously described [32, 33] and transplanted into lethally irradiated mice 3 to 4 hours prior to transplantation together with

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300,000 wBM competitor cells. WBM transplantations were performed using cell numbers obtained from 1/3 of a bone or 1-5x106 cells. Cells were injected into the tail-vein of recipient animals. The test cell-derived contribution to the

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different peripheral blood cell lineages was followed for at least 16 weeks,

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unless otherwise indicated, to assess the long-term contribution of the transplanted HSCs. Analysis

Bone marrow. HSCs were defined as LSK CD48-CD150+, isolated and

analyzed as previously described [32, 33]. Peripheral blood. PB analyzes were conducted as previously described [32] and analyzed on an LSRII (BD Biosciences).

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ACCEPTED MANUSCRIPT Statistics Results were statistically analyzed and figures prepared using Excel and GraphPad Prism (GraphPad Inc.) software. Two-tailed Student’s t tests were

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as follows: *p < 0.05, **p < 0.001, and ***p < 0.0001.

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used throughout to evaluate statistical significance. Significance is indicated

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ACCEPTED MANUSCRIPT AUTHOR CONTRIBUTIONS

A.R.N. designed and performed experiments, analyzed data, and wrote the paper. D.B. and C.J.P. designed experiments, conceived and supervised the

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study, and wrote the paper.

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ACKNOWLEDGMENTS

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We thank Gerd Sten for technical support and laboratory maintenance. This work was supported by grants to D.B. from the Swedish Research Council, Cancerfonden and ERC Consolidator grant, and to C.J.P. by grants from the Swedish

Research

Council,

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(Avtal

om

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TABLE LEGENDS

and

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Läkarutbildning och Forskning).

Barncancerfonden

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Table 1. Variation in reconstitution levels of separate bones in primary and secondary recipients. PB granulocyte and BM HSC chimerism levels in tibia-, femur- and hipbones of 36 individual mice. Recipient mice represent a mixture of mice, primary and secondary transplanted, with varying numbers of sorted HSCs or wBM test cells. wBM from separate bones of three mice indicated with asterisks were serially transplanted to functionally assess HSC content. PB granulocyte chimerism levels of secondary recipients of the separate bones are indicated in rows below donor mice, in the same column

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ACCEPTED MANUSCRIPT as the donor bone. The test cell-derived contribution was evaluated at least 16 weeks post transplantation in all mice except for #4, 15 and 19, which were

SUPPLEMENTARY TABLE LEGENDS

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analyzed 12 weeks post transplantation.

Supplementary table 1. Variation in reconstitution levels of separate

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bones in primary and secondary recipients. Overall PB, PB granulocyte and BM HSC chimerism levels in tibia-, femur- and hipbones of 36 individual

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mice. Recipient mice represent a mixture of mice, primary and secondary transplanted, with varying numbers of sorted HSCs or wBM test cells. Cellular source indicates the purity of the transplanted test cells. wBM from separate bones of three mice indicated with asterisks were serially transplanted to

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functionally assess HSC content. PB granulocyte chimerism levels of secondary recipients of the separate bones are indicated in rows below donor mice, in the same column as the donor bone. The test cell-derived

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contribution was evaluated at least 16 weeks post transplantation in all mice

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except for #4, 15 and 19, which were analyzed 12 weeks post transplantation.

FIGURE LEGENDS

1. Variations caused by transplantation method. (A) Gating strategy for hematopoietic stem cells. Pre-gated on viable singlets. Lineage distribution among donor-derived cells in mice primary (B) or serially (C) transplanted with sorted HSCs or wBM. Primary HSC recipients n = 25, primary wBM recipients

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ACCEPTED MANUSCRIPT n = 25, secondary HSC recipients n = 34, secondary wBM recipients n = 45. (D) PB granulocyte chimerism levels 12 weeks post transplantation of mice serially transplanted with HSCs together with either fresh or previously transplanted competitor cells. Error bars depict means ± SEM. Analyses were

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done with unpaired Students’s t-tests. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2. Experimental strategy for serial transplantation. Primary

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transplantation with wBM, maximizing the numbers of isolable HSCs for serial transplantation. BM is subsequently extracted from multiple bones from

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primary recipient mice to obtain the most representative clonal variation of the host’s HSC pool. The same numbers of HSCs are then sorted and serially

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transplanted with fresh or previously transplanted wBM competitor cells.

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topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell,

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Table 1

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44.3 47.2 50.6 50.7 51.8

Mouse #18 Mouse #19 Mouse #20 Mouse #21 Mouse #22 Mouse #23 Mouse #24 Mouse #25 Mouse #26 Mouse #27 Mouse #28 Mouse #29 Mouse #30 Mouse #31 Mouse #32 Mouse #33 Mouse #34 Mouse #35 Mouse #36

63.7 69.4 75.0 75.6 75.8 81.9 82.2 83.9 86.9 87.5 88.6 89.1 89.5 91.5 91.8 94.8 95.0 95.7 100.0

femur

tibia

4.7 5.7 35.5 3.8 2.4 8.6 88.5 76.6 3.5 5.7 0.9 0.5 1.6 30.8 13.5/0.0 27.7/8.7 28.1 57.4 24.7 26.4 3.6 13.5 4.2 7.3 7.9 13.8 5.5/na 20.2/na 41.5 54.1 0.2 1.4 93.4 94.8 34.4 75.0 53.9 13.0 61.1/36.0 0.9/1.8 17.0 7.0 82.8 76.3 87.4 65.8 41.2 28.6 84.6 70.7 83.0 94.5 39.6 32.1 12.1 87.1 38.1 96.1 95.5 66.0 95.5 99.4 65.0 67.3 87.7 95.6 80.4 92.2 90.5 85.8 92.1 90.6 92.3 88.5 26.7 57.9 28.6 28.6

na 6.1 6.4 na 12.5 18.0 2.9 0.5/na 82.1 na 42.5 na 10.7 1.7/7.0 52.0 2.7 na na 28.0 1.8/0.8 na na 78.5 na na 83.0 na na na na 98.2 56.1 90.7 na na na na na na

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Mouse #13 Mouse #14 Mouse #15 Mouse #16 Mouse #17***

na 5.5 2.6 12.3 31.4 7.3 5.4 1.5 8.8 na 70.1 59.0 22.2 12.5 42.4 0.0 0.0 0.0 0.0 75.6 1.2 9.1/0.1 95.6/59.3 1.0/2.4 42.9 66.7 95.4 na 13.6 13.8 7.7 13.3 4.5 na 2.1 2.5 4.7 2.6 1.9 0.2/20.6 7.6/1.6 3.4/2.4 76.2 61.1 41.7 3.6 0.4 0.4 na 44.4 85.4 na 18.2 66.7 11.6 8.3 1.2 0.0/0.0 0.0/0.1 0.4/0.5 na 18.8 0.0 na 77.2 75.3 85.2 60.2 47.1 na 32.0 40.0 na 69.2 71.4 78.7 98.4 65.0 na 50.0 54.3 na 5.3 70.9 na 5.3 56.5 na 54.0 74.3 98.7 na 98.4 65.8 82.0 55.1 99.1 98.9 92.6 na 85.7 84.5 na 86.0 41.0 na 71.5 79.6 na 81.0 61.5 na 17.9 72.1 na 30.8 79.2

hip

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Mouse #8 Mouse #9 Mouse #10 Mouse #11 Mouse #12**

femur

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0.1 1.6 2.2 13.4 15.7 17.3 25.3

tibia

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Mouse #1 Mouse #2 Mouse #3 Mouse #4 Mouse #5 Mouse #6 Mouse #7*

Right HSC chimerism (%)

Left HSC chimerism (%)

Granulocyte PB chimerism

Figure 1

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a

102 0

10

3

102 0

103 104 : Lin

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103 104 : CD150

CD150

b

c

4

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HSCs

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103 104 : Sca-1

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1°transplantation

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Lineage

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Competitor cells

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c-kit

10

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Figure 2

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Secondary transplantation

Isolation of bone marrow Multiple bones to minimize influence of variances in HSC distribution

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Primary transplantation

Sorted HSCs to allow for functional evaluation on a per cell basis and ensure that all donor-derived cells are the progeny of the serially transplanted HSCs

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wBM to ensure robust reconstitution and maximize selfrenewal

ACCEPTED Left HSCMANUSCRIPT 
 Right HSC 
 chimerism (%)" chimerism (%)"

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!! Mouse!#1! Mouse!#2! Mouse!#3! Mouse!#4! Mouse!#5! Mouse!#6! Mouse!#7*! !! Mouse!#8! Mouse!#9! Mouse!#10! Mouse!#11! Mouse!#12**! !! Mouse!#13! Mouse!#14! Mouse!#15! Mouse!#16! Mouse!#17***! !! Mouse!#18! Mouse!#19! Mouse!#20! Mouse!#21! Mouse!#22! Mouse!#23! Mouse!#24! Mouse!#25! Mouse!#26! Mouse!#27! Mouse!#28! Mouse!#29! Mouse!#30! Mouse!#31! Mouse!#32! Mouse!#33! Mouse!#34! Mouse!#35! Mouse!#36!

Overall" Granulocyte" PB " PB " ! ! "bia! femur! hip! ! chimerism" chimerism" hip! femur! "bia! ! ! 1.9! 0.1! ! na! 5.5! 2.6! ! 4.7! 5.7! na! ! 0.4! 1.6! ! 12.3! 31.4! 7.3! ! 35.5! 3.8! 6.1! ! 3.3! 2.2! ! 5.4! 1.5! 8.8! ! 2.4! 8.6! 6.4! ! 37.8! 13.4! ! na! 70.1! 59.0! ! 88.5! 76.6! na! ! 34.5! 15.7! ! 22.2! 12.5! 42.4! ! 3.5! 5.7! 12.5! ! 66.0! 17.3! ! 0.0! 0.0! 0.0! ! 0.9! 0.5! 18.0! ! 47.0! 25.3! ! 0.0! 75.6! 1.2! ! 1.6! 30.8! 2.9! ! ! ! ! 9.1/0.1! 95.6/59.3! 1.0/2.4! ! 13.5/0.0! 27.7/8.7! 0.5/na! ! 10.1! 29.5! ! 42.9! 66.7! 95.4! ! 28.1! 57.4! 82.1! ! 21.7! 34.8! ! na! 13.6! 13.8! ! 24.7! 26.4! na! ! 31.0! 36.7! ! 7.7! 13.3! 4.5! ! 3.6! 13.5! 42.5! ! 55.9! 37.2! ! na! 2.1! 2.5! ! 4.2! 7.3! na! ! 35.7! 38.1! ! 4.7! 2.6! 1.9! ! 7.9! 13.8! 10.7! ! ! ! ! 0.2/20.6! 7.6/1.6! 3.4/2.4! ! 5.5/na! 20.2/na! 1.7/7.0! ! 46.1! 44.3! ! 76.2! 61.1! 41.7! ! 41.5! 54.1! 52.0! ! 21.4! 47.2! ! 3.6! 0.4! 0.4! ! 0.2! 1.4! 2.7! ! 44.2! 50.6! ! na! 44.4! 85.4! ! 93.4! 94.8! na! ! 44.7! 50.7! ! na! 18.2! 66.7! ! 34.4! 75.0! na! ! 39.6! 51.8! ! 11.6! 8.3! 1.2! ! 53.9! 13.0! 28.0! ! ! ! ! 0.0/0.0! 0.0/0.1! 0.4/0.5! ! 61.1/36.0! 0.9/1.8! 1.8/0.8! ! 35.3! 63.7! ! na! 18.8! 0.0! ! 17.0! 7.0! na! ! 68.1! 69.4! ! na! 77.2! 75.3! ! 82.8! 76.3! na! ! 76.5! 75.0! ! 85.2! 60.2! 47.1! ! 87.4! 65.8! 78.5! ! 27.9! 75.6! ! na! 32.0! 40.0! ! 41.2! 28.6! na! ! 83.9! 75.8! ! na! 69.2! 71.4! ! 84.6! 70.7! na! ! 67.3! 81.9! ! 78.7! 98.4! 65.0! ! 83.0! 94.5! 83.0! ! 65.1! 82.2! ! na! 50.0! 54.3! ! 39.6! 32.1! na! ! 89.0! 83.9! ! na! 5.3! 70.9! ! 12.1! 87.1! na! ! 57.6! 86.9! ! na! 5.3! 56.5! ! 38.1! 96.1! na! ! 71.5! 87.5! ! na! 54.0! 74.3! ! 95.5! 66.0! na! ! 39.9! 88.6! ! 98.7! na! 98.4! ! 95.5! 99.4! 98.2! ! 77.0! 89.1! ! 65.8! 82.0! 55.1! ! 65.0! 67.3! 56.1! ! 76.6! 89.5! ! 99.1! 98.9! 92.6! ! 87.7! 95.6! 90.7! ! 57.0! 91.5! ! na! 85.7! 84.5! ! 80.4! 92.2! na! ! 82.1! 91.8! ! na! 86.0! 41.0! ! 90.5! 85.8! na! ! 93.7! 94.8! ! na! 71.5! 79.6! ! 92.1! 90.6! na! ! 94.1! 95.0! ! na! 81.0! 61.5! ! 92.3! 88.5! na! ! 94.8! 95.7! ! na! 17.9! 72.1! ! 26.7! 57.9! na! ! 92.5! 100.0! ! na! 30.8! 79.2! ! 28.6! 28.6! na! !!

Cellular " Recipient" source" ! ! 2°# wBM! 2°# wBM! 2°# wBM! HSCs# 1°# 1°# wBM! 2°# wBM! 1°# wBM! wBM! wBM! wBM! wBM! wBM!

2°# 2°# 1°# 2°# 1°#

wBM! wBM! HSCs# wBM! wBM!

1°# 2°# 1°# 2°# 1°#

wBM! HSCs# wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM! wBM!

2°# 1°# 1°# 2°# 1°# 1°# 2°# 1°# 2°# 2°# 1°# 1°# 1°# 2°# 2°# 1°# 1°# 1°# 2°#