Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Sep 29:12:20417314211044855.
doi: 10.1177/20417314211044855. eCollection 2021 Jan-Dec.

Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells

Gordian Born  1   2 , Marina Nikolova  3 , Arnaud Scherberich  1   2 , Barbara Treutlein  3 , Andrés García-García  1 , Ivan Martin  1   2
Affiliations

Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells

Gordian Born et al. J Tissue Eng. .

Abstract

Hematopoietic stem and progenitor cells (HSPCs) are frequently located around the bone marrow (BM) vasculature. These so-called perivascular niches regulate HSC function both in health and disease, but they have been poorly studied in humans due to the scarcity of models integrating complete human vascular structures. Herein, we propose the stromal vascular fraction (SVF) derived from human adipose tissue as a cell source to vascularize 3D osteoblastic BM niches engineered in perfusion bioreactors. We show that SVF cells form self-assembled capillary structures, composed by endothelial and perivascular cells, that add to the osteogenic matrix secreted by BM mesenchymal stromal cells in these engineered niches. In comparison to avascular osteoblastic niches, vascularized BM niches better maintain immunophenotypically-defined cord blood (CB) HSCs without affecting cell proliferation. In contrast, HSPCs cultured in vascularized BM niches showed increased CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) numbers. The vascularization also contributed to better preserve osteogenic gene expression in the niche, demonstrating that niche vascularization has an influence on both hematopoietic and stromal compartments. In summary, we have engineered a fully humanized and vascularized 3D BM tissue to model native human endosteal perivascular niches and revealed functional implications of this vascularization in sustaining undifferentiated CB HSPCs. This system provides a unique modular platform to explore hemato-vascular interactions in human healthy/pathological hematopoiesis.

Keywords: Hematopoietic stem cell; bone marrow microenvironment; engineered 3D niches; human hematopoiesis; perivascular niche.

PubMed Disclaimer

Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Experimental design illustrating the generation of engineered niches within perfusion bioreactors. Avascular osteoblastic BM niches (upper row) were engineered by seeding bone marrow mesenchymal stromal cells (BM-MSCs) in hydroxyapatite-based EngiPore scaffolds. Cells were first cultured for 7 days in proliferative medium and then for another 21 days in osteogenic medium (total 28 days). To engineer vascularized BM niches (bottom row), stromal vascular fraction (SVF) cells were seeded in niches engineered with BM-MSCs after 1 week of osteogenic differentiation (day 14) and cultured for 2 weeks in osteogenic medium plus FGF2 (total 28 days). See materials and methods for detailed mediums composition. At final analysis, engineered niches were harvested and processed for Q-PCR, flow activated cell sorting (FACS) and histology.
Figure 2.
Figure 2.
Vascular structures are found only in engineered niches including SVF cells. (a–f) Whole-mount immunostaining for CD31 (red) and VEGF (green) in niches engineered with only BM-MSCs (a–c) or BM-MSCs + SVF cells (d–f). (c, f) Nuclei are labeled with DAPI (blue). Scale bar, 100 µm. (g) VEGF protein content in engineered niches. (h, i) Q-PCR data showing ANGPT-1 and VCAM1 gene expression in engineered niches. Data are plotted as means ± standard deviations; n = 4–12; **p < 0.01, ***p < 0.001. Unpaired two-tailed t tests.
Figure 3.
Figure 3.
SVF-induced vascularization does not compromise niche osteogenic matrix. (a–f) Whole-mount immunostaining for COL1A1 (red) and OCN (green) in niches engineered with only BM-MSCs (a–c) or BM-MSCs + SVF cells (d–f). (c, f) Nuclei are labeled with DAPI (blue). Scale bar, 100 µm. (g, h) Q-PCR data showing ALPL and SP7 gene expression in engineered niches. Data are plotted as means ± standard deviations; n = 4–12. Unpaired 2-tailed t tests.
Figure 4.
Figure 4.
Vascularized BM niches preserve undifferentiated CB HSCs better than avascular BM niches. (a) Experimental setup depicting the 1-week culture of CB HSPCs in engineered niches and the different read-outs performed in the supernatant (SN) and niche compartments. (b–g) Percentage of (b, e) CD45+ cells. (c–f) CD34+CD38−CD45RA−CD90+ HSCs and (d, g) CD34−CD38− mature cells retrieved from the SN and niche compartments at final analysis. (h–k) Whole-mount immunostaining for CD31 (red), NG2 (green) and CD34 (gray) in BM niches vascularized with SVF cells. (k) Nuclei are labeled with DAPI (blue). Scale bar, 50 µm, and (b–g) data are plotted as means ± standard deviations; n = 6–8. **p < 0.01. Unpaired two-tailed t tests.
Figure 5.
Figure 5.
Vascularized BM niches does not impact the proliferation of CB HSCs, but affect their differentiation potential. (a, b) Q-PCR data showing CD164 and Ki67 gene expression in HSCs sorted from engineered BM niches. (c) Hoechst 42/Ki67 staining of HSCs sorted after 1-week coculture in engineered niches. Ratio G1/G0 is plotted as an indicator of the proliferative status. (d, e) Fold increase in (d) total colony forming-units in culture (CFU-C) and (e) colony forming-units granulocytes-erythrocytes-monocytes-megakaryocytes (CFU-GEMM) normalized to uncultured CD34+CD38− cells. (f, g) Representative images showing CFU-GMM from (f) avascular and (g) vascularized niches. (a–e) Data are plotted as means ± standard deviations; n = 4; *p < 0.05. Unpaired two-tailed t tests.
Figure 6.
Figure 6.
SVF-induced vascularization contributes to maintain the osteogenic potential of engineered BM niches. (a–c) Percentage of (a) CD45−CD31+CD146− endothelial cells and (b) CD45−CD31−CD146+ perivascular cells and (c) CD45−CD31−CD146−CD90+ mesenchymal stromal cells in engineered niches after 1-week coculture with CB HSPCs. (d–f) Fold change gene expression of SP7, OCN, and ALPL in sorted MSCs after the 1-week coculture with/without CB HSPCs. Data are plotted as means ± standard deviations; n = 6–8. *p < 0.05, **p < 0.01, ***p < 0.001. (a–c) Unpaired two-tailed t tests. (d–f) One-way ANOVA with Tukey’s multiple comparison tests.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Morrison SJ, Scadden DT.The bone marrow niche for haematopoietic stem cells. Nature 2014; 505(7483): 327–334. - PMC - PubMed
    1. Pinho S, Frenette PS.Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol 2019; 20(5): 303–320. - PMC - PubMed
    1. Méndez-Ferrer S, Michurina TV, Ferraro F, et al.. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010; 466(7308): 829–834. - PMC - PubMed
    1. Boulais PE, Frenette PS.Making sense of hematopoietic stem cell niches. Blood 2015; 125(17): 2621–2629. - PMC - PubMed
    1. Ramasamy SK, Kusumbe AP, Itkin T, et al.. Regulation of hematopoiesis and osteogenesis by blood vessel-derived signals. Annu Rev Cell Dev Biol 2016; 32: 649–675. - PubMed

LinkOut - more resources