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Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche

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Abstract

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the ‘niche’, within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method – ‘whole bone subcutaneous transplantation’- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

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Data Availability

Data will be made available on reasonable request.

Abbreviations

BM:

Bone Marrow

BSA:

Bovine Serum Albumin

CAR:

Abundant Reticular Stromal cells

Cas9:

CRISPR-Associated protein 9

CD:

Cluster Differentiation

CEUA:

Ethics Animal Care and Use Committee

CRE-ER:

Cre-Estrogen Receptor (ER)

CRISPR:

Clustered Regularly Interspaced Short Palindromic Repeats

CXCL12:

Chemokine (C-X-C motif) Ligand 12

DMEM:

Dulbecco’s Modified Eagle Mediu

DT:

Diphtheria Toxin

FBS:

Fetal Bovine Serum

FSC-A:

Forward Scatter Area

FSC-H:

Forward Scatter Height

GFP:

Green Fluorescent Protein

HSC:

Hematopoietic Stem Cells

iDTR:

inducible Diphtheria Toxin Receptor

LKS:

Lineage (Lin)− Sca-1+ c-Kit+

Myh11:

Myosin Heavy Chain 11

NK:

Natural Killer

NOD/SCID:

Nonobese Diabetic/Severe Combined Immunodeficiency

OCT:

Tissue-Tek

PBS:

Phosphate-Buffered Saline

PCR:

Polymerase Chain Reaction

PFA:

Paraformaldehyde

SEM:

Standard Error

TH:

Tyrosine Hydroxylase

UFMG:

Federal University of Minas Gerais

WT:

Wild-Type

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Acknowledgements

Alexander Birbrair is supported by a research productivity fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PQ2), a grant from Instituto Serrapilheira/Serra-1708-15285, a grant from Pró-reitoria de Pesquisa/Universidade Federal de Minas Gerais (PRPq/UFMG) (Edital 05/2016); a grant from Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG (Chamada N°01/2021 – Demanda Universal, APQ-01321-21); a grant from FAPEMIG [Rede Mineira de Pesquisa Translacional em Imunobiológicos e Biofármacos no Câncer (REMITRIBIC, RED-00031-21)]; a grant from FAPEMIG [Rede Mineira de Engenharia de Tecidos e Terapia Celular (REMETTEC, RED-00570-16)]; a grant from FAPEMIG [Rede De Pesquisa Em Doenças Infecciosas Humanas E Animais Do Estado De Minas Gerais (RED-00313-16)]; and a grant from MCTIC/CNPq Nº 28/2018 (Universal/Faixa A). Akiva Mintz is supported by the National Institute of Health (1R01CA179072-01A1) and by the American Cancer Society Mentored Research Scholar grant (124443-MRSG-13-121-01-CDD). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, as Thamires Righi was supported by a PrInt/CAPES fellowship. Caroline C. Picoli was supported by doctoral fellowships from CAPES. Patricia R. Martins was supported by a postdoctoral fellowship (PDJ) from CNPq. The authors also thank CAPI (UFMG) for microscopical technical support and Laboratory of Flow Cytometry at the Instituto de Ciências Biológicas/UFMG (http://labs.icb.ufmg.br/citometria/)” for providing the equipment and technical support for experiments involving flow cytometry.

Funding

Alexander Birbrair is supported by a research productivity fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PQ2), a grant from Instituto Serrapilheira/Serra-1708-15285, a grant from Pró-reitoria de Pesquisa/Universidade Federal de Minas Gerais (PRPq/UFMG) (Edital 05/2016); a grant from Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG (Chamada N°01/2021 – Demanda Universal, APQ-01321-21); a grant from FAPEMIG [Rede Mineira de Pesquisa Translacional em Imunobiológicos e Biofármacos no Câncer (REMITRIBIC, RED-00031-21)]; a grant from FAPEMIG [Rede Mineira de Engenharia de Tecidos e Terapia Celular (REMETTEC, RED-00570-16)]; a grant from FAPEMIG [Rede De Pesquisa Em Doenças Infecciosas Humanas E Animais Do Estado De Minas Gerais (RED-00313-16)]; and a grant from MCTIC/CNPq Nº 28/2018 (Universal/Faixa A). Akiva Mintz is supported by the National Institute of Health (1R01CA179072-01A1) and by the American Cancer Society Mentored Research Scholar grant (124443-MRSG-13-121-01-CDD). Caroline C. Picoli was supported by doctoral fellowships from CAPES. Patricia R. Martins was supported by a postdoctoral fellowship (PDJ) from CNPq. The authors also thank CAPI (UFMG) for microscopical technical support and Laboratory of Flow Cytometry at the Instituto de Ciências Biológicas/UFMG (http://labs.icb.ufmg.br/citometria/)” for providing the equipment and technical support for experiments involving flow cytometry.

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Authors and Affiliations

  1. Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

    Caroline C. Picoli, Patrícia Rocha Martins & Alexander Birbrair

  2. Department of Dermatology, University of Wisconsin-Madison, Medical Sciences Center, Rm 4385, 1300 University Avenue, Madison, WI, 53706, USA

    Xiao Lin Casey Wong, Thamires Righi, Alexandre Kanashiro & Alexander Birbrair

  3. Department of Pharmacology, Federal University of São Paulo, São Paulo, SP, Brazil

    Thamires Righi & Fabio Cardoso Cruz

  4. Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

    Pedro P. G. Guimarães

  5. Institute of Biological Sciences, Federal University of Goiás, Goiânia, GO, Brazil

    Mauro C. X. Pinto

  6. Center of Biological Sciences and Health, Federal University of West Bahia, Barreiras, BA, Brazil

    Jaime H. Amorim

  7. Department of Genetics, Ecology and Evolution, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

    Vasco A. C. Azevedo

  8. Department of Biology, Federal University of Maranhão, São Luís, MA, Brazil

    Silma Regina Pereira

  9. Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

    Rodrigo R. Resende

  10. Department of Radiology, Columbia University Medical Center, New York, NY, USA

    Akiva Mintz & Alexander Birbrair

  11. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York, NY, USA

    Paul S. Frenette

  12. Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Paul S. Frenette

  13. Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

    Paul S. Frenette

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  1. Caroline C. Picoli
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Contributions

AB conceived and supervised the study; CCP, PRM, XLCW, TR, PPGG, MCXP, JHA, VACA, SRP, AK, FCC, RRR, AM, AB analyzed the data and discussed the results; AB and PSF were responsible for funding; AM and AB wrote the original draft; all authors contributed to and approved the final version of the manuscript.

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Correspondence to Alexander Birbrair.

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Caroline C. Picoli and Patrícia Rocha Martins contributed equally and were co-first authors in this manuscript.

Supplementary Information

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Supplementary Fig. 1

Transplantation of whole bone with the surrounding skeletal muscle does not improve the amount of hematopoietic stem cells in the subcutaneously transplanted bones. A C57BL6-derived femur with surrounding skeletal muscle subcutaneous transplantation into recipient NOD/SCID mice scheme. Whole femurs with muscle from C57BL6 mouse were implanted subcutaneously into NOD/SCID mice. After 45 days, the subcutaneously transplanted femurs from donor C57BL6 were collected for analysis. B Representative flow cytometry dot plots of bone marrow cells are shown. Data were assessed by flow cytometry analysis of bone marrow cells isolated from the femurs. Representative flow cytometry plots of femurs with surrounding skeletal muscle from C57BL6 mice implanted subcutaneously into C57BL6 mice analyzed after 45 days. The gating strategy was applied to analyze LSK (Sca-1 + c-kit + Lineage − gated). (PNG 429 KB)

High Resolution Image (TIF 962 KB)

Supplementary Fig. 2

Transplantation of whole bone with the surrounding skeletal muscle does not improve the amount of hematopoietic stem cells in the subcutaneously transplanted bones. A C57BL6-derived femur with surrounding skeletal muscle subcutaneous transplantation into recipient NOD/SCID mice scheme. Whole femurs with muscle from C57BL6 mouse were implanted subcutaneously into NOD/SCID mice. After 45 days, the subcutaneously transplanted femurs from donor C57BL6 were collected for analysis. B Representative flow cytometry dot plots of bone marrow cells are shown. Data were assessed by flow cytometry analysis of bone marrow cells isolated from the femurs. Representative flow cytometry plots of femurs with surrounding skeletal muscle from C57BL6 mice implanted subcutaneously into C57BL6 mice analyzed after 45 days. The gating strategy was applied to analyze LSK (Sca-1 + c-kit + Lineage − gated). (PNG 4.16 MB)

High Resolution Image (TIF 5.08 MB)

Supplementary Fig. 3

Subcutaneously transplanted bones lack sympathetic innervation. A Schematic diagram of femurs derived from Myh11CreER/TdTomato mice subcutaneously transplanted into C57BL6 WT mice. Femurs from Myh11CreER/TdTomato mice were dissected and transplanted subcutaneously into recipient C57BL6 WT mice. After 3 months, the animals were submitted to tamoxifen treatment for 5 days, and, after 2 days, the subcutaneously transplanted femurs were collected for analyses. B Representative photomicrograph of a femur section showing Myh11CreER+/TdTomato+ peri-arteriolar cells (red), CD31+/CD144+ blood vessels (blue), TH+ sympathetic nerve fibers (green) and DAPI+ nuclei (white). (PNG 3.05 MB)

High Resolution Image (TIF 4.53 MB)

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Picoli, C.C., Martins, P.R., Wong, X.L.C. et al. Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche. Stem Cell Rev and Rep 19, 906–927 (2023). https://doi.org/10.1007/s12015-022-10496-9

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