In vitro biomimetic engineering of a human hematopoietic niche with functional properties

Three-Dimensional Microenvironments Can Be Engineered Within the Perfusion Bioreactor System.

The generation of the 3D microenvironments was performed by differentiation of primary BM-derived hMSCs on ceramic materials within a perfusion bioreactor. Cells were first labeled with a VENUS transgene (>93%) (SI Appendix, Fig. S1A) to facilitate subsequent analysis. To achieve the engineering of an osteoblastic-like stroma, we adopted a protocol previously used for the generation of bone grafts (32) (Fig. 1A). hMSCs were first cultured 1 wk in proliferative medium (PM) to increase cell number and ensure scaffold colonization, followed by 3 wk of osteogenic medium (OM) supplementation, to promote cell differentiation while stimulating extracellular matrix (ECM) production (33). The resulting tissue was defined as “engineered niche” (eN) (Fig. 1A) while naked ceramic (Ce) (Fig. 1A), not containing hMSCs but loaded with CD34⁺ cells, was used as internal 3D culture control. The blood compartment was subsequently introduced by injecting CB-derived human CD34⁺ cells from single donors into the device tubing. The in vitro coculture was sustained for 1 wk in serum-free medium supplemented with a low concentration of hematopoietic cytokines [10 ng/mL thrombopoietin (TPO), stem cell factor (SCF), and Fms-related tyrosine kinase 3 ligand (Flt3-L)]. Upon retrieval from the bioreactor chamber, the Ce was devoid of apparent ECM structures (Ce, SI Appendix, Fig. S1B) while the eN exhibited features of an engineered tissue with thick gel-like structures homogenously covering the starting material (eN, Fig. 1B). Scanning electron microscopy confirmed the deposition of an ECM which embeds cells, presumably of both stromal (fibroblastic shape, Fig. 1B) and blood origins (round shape, Fig. 1B), including dividing cells (Fig. 1B).

Engineered 3D Microenvironments Allow the Maintenance of Hematopoietic Stem and Progenitor Cells with Functional Properties.

To characterize their cellular composition, the 3D microenvironments were digested for cell retrieval (>92% retrieval efficiency, with an overall cell death below 1.5%) (SI Appendix, Fig. S1 B and C) and subsequent quantitative phenotypic analysis (SI Appendix, Fig. S1 D and E).

The eN was composed of over 4.7 × 10⁶ blood cells (SI Appendix, Fig. S1F) per bioreactor system [61-fold increase (f.i.) over the initial 70,000 CD34⁺ cells] (SI Appendix, Fig. S1G). In contrast, in the absence of engineered stroma, blood cell expansion was limited to a 7.8 f.i. (SI Appendix, Fig. S1 F and G). With a ratio of HSPCs over committed cells (CD34⁻/CD38⁺) lower than the Ce (38 vs. 204) (SI Appendix, Fig. S1H), the eN was shown to sustain the proliferation of differentiated populations. The eN also promoted the maintenance of phenotypic hematopoietic stem and progenitor populations (HSPCs) (Fig. 2A), yielding systematically higher total numbers of HSPCs (46.2 f.i. vs. 6.4 for Ce), including HSCs (13.8 f.i. vs. 1.6), multipotent progenitors (MPPs) (8.5 f.i. vs. 2.3), and multipotent lymphoid progenitors (MLPs) (161 f.i. vs. 20) (SI Appendix, Fig. S2A).

The capacity of hMSCs to support the proliferation of CD34⁺ cells was further confirmed in a 2D setup (SI Appendix, Fig. S2B). Using undifferentiated hMSCs as feeder layer (2D hMSCs) compared with a hMSC-free control (2D), we observed a significant increase of phenotypic HSPCs (100 f.i. vs. 15), HSCs (29 f.i. vs. 4), MPPs (43 f.i. vs. 16), and MLPs (279 f.i. vs. 21) (SI Appendix, Fig. S2B), in line with results derived from well-established Dexter cultures (34, 35). The capacity of the generated stroma to preserve the functionality of cultured blood cells was assessed both by in vitro and in vivo assays. The corresponding donor cells before in vitro culture (“uncultured”) were used as functional positive control. In vitro colony-forming unit assays demonstrated the maintenance of stem or progenitor cell capacities (with multilineage and proliferation potential) of cultured CD34⁺ cells, indicated by the generation of myeloid colonies with the same morphology as their uncultured counterparts (SI Appendix, Fig. S2C). Colony numbers were increased for cells retrieved from 3D cultures for colony-forming unit-granulocyte and macrophage (GM) (42.8 f.i vs. 4.8 for Ce), burst-forming unit-erythroid (BFU-E) (113 f.i vs. 18), and Colony-forming unit-Granulocyte (GEmM) (36.5 f.i vs. 4.8) colonies (Fig. 2B).

Stem and progenitor potential of cultured cells was further tested by intrafemoral transplantation of equal numbers of CD34⁺ cells from Ce or eN in irradiated NSG mice. The human blood compartment was successfully reconstituted in all groups [>1% human CD45 positive cells (hCD45) in peripheral blood] (Fig. 2C). Chimerism was detectable as early as week 6 and persisted over 28 wk posttransplantation, indicating the long-term repopulation capacity of transplanted cells. As anticipated, the highest chimerism was obtained by transplantation of uncultured CD34⁺ serving as positive control (27.9 ± 3.1%) (Fig. 2C). CD34⁺ cells derived from Ce and eN exhibited similar reconstitution levels, with an average of 2.7 ± 0.8% and 6.4 ± 1% of hCD45 respectively, detected in the peripheral blood (Fig. 2C). The chimerism was also assessed in the BM and the spleen of animals with human cells robustly engrafted in all conditions, with a trend for higher hCD45 frequency detected in the eN than in the Ce group, both in BM (6.4% ± 0.5 vs. 5.2% ± 2.5, Fig. 2D) and the spleen (12.6% ± 4.4 vs. 5.6% ± 2.8, Fig. 2D). Functionality of CD34⁺ cells was further assessed by multilineage reconstitution capacity. At week 18 posttransplantation, cells from all conditions successfully reconstituted the myeloid and lymphoid lineages (SI Appendix, Fig. S2D).

These data demonstrate that, compared with the Ce counterparts, the eN yields hematopoietic cells with similar reconstitution potential, but in higher numbers. This suggests that the generated tissue provides cues capable to enhance the maintenance/expansion of CFU-HSPCs with in vivo engraftment and multilineages reconstitution potential.

Molecular Characterization of the Engineered Niche Reveals the Establishment of an Osteoblastic-Like 3D Environment.

To identify factors associated with robust hematopoiesis development, the eN was further characterized. First, we monitored the secretion of key cytokines throughout culture times. Inflammatory factors [interleukin 6 (IL-6), interleukin 8 (IL-8), macrophage colony-stimulating factor (MCSF) (Fig. 3A), and monocyte chemoattractant protein-1 (MCP-1)] showed the highest differences and were found at high concentrations in eN. In particular, IL-6 and IL-8 production increased substantially upon HSPC addition (eN, Fig. 3A). Flt3-L, TPO, and SCF proteins were found at concentrations similar to those supplemented in the coculture medium (SI Appendix, Fig. S3A), suggesting that hMSCs secrete low levels of those HSPC supportive factors. Vascular endothelial growth factor α (VEGFα) and angiopoietin 1 (Ang-1) were also detected at significant levels (eN, SI Appendix, Fig. S3A) although Ang-1 decreased over time to remain stable after HSPC loading.

To obtain a more comprehensive understanding of the cellular compartments associated with factor secretion, we isolated both blood progenitors (CD34⁺) and mesenchymal populations before (defined as hMSC day 28) (SI Appendix, Fig. S3B) and after CD34⁺ coculture (defined as eN-hMSC day 35, SI Appendix, Fig. S3B). This confirmed the strong expression of inflammatory cytokines (IL-6, MCSF) by hMSCs during coculture with HSPCs, with levels markedly higher than those in blood progenitor cells (Fig. 3B). IL-8 and MCP-1 were expressed by both blood and mesenchymal cells (Fig. 3B). Interestingly, following coculture with CD34⁺ cells, hMSCs substantially increased their IL-6 and IL-8 expression (Fig. 3B).

To assess the role of IL-6 and IL-8 in the system, we investigated the effect of their addition in the Ce condition (SI Appendix, Fig. S4A). IL-6 and IL-8 at doses corresponding to those measured in the eN led to a significant increase in the number of HSPCs, committed progenitors (CD34⁺/CD38⁺), Granulocyte-monocytes progenitors (GMPs), and MLPs (SI Appendix, Fig. S4B). However, no effect was measured on HSCs and MPPs (SI Appendix, Fig. S4B). This suggests that these inflammatory cytokines mediate the proliferation of committed populations in the eN whereas the observed stem cell compartment expansion is driven by other hMSC factors.

Before CD34⁺ loading, hMSCs in the engineered tissue predominantly consisted of not only osteoblastic-like cells, but also of a pool expressing progenitor markers (SI Appendix, Fig. S5). We further analyzed the transcription profile of eN-hMSCs as niche cells at the end of the culture (eN-hMSC day 35) and compared it to postexpanded hMSCs (hMSC Day 0) and hMSCs in the eN before CD34⁺ loading (hMSC day 28). This confirmed the osteoblastic profile of hMSC (day 35) at the end of the 3D culture, evidenced by the up-regulation of alkaline phosphatase (ALP) and bone sialoprotein (BSP) (17 and 869 f.i., respectively) (Fig. 3C). Interestingly, a marked increase in Nestin expression (117 f.i.) was acquired by hMSCs after coculture with blood cells (Fig. 3C), as well as a decrease in hypoxia-inducible factor 1-alpha (HIF1α, 8.9-fold) expression.

Thus, by combining protein and gene expression analyses, we describe an osteoblastic (36, 37) and niche-associated (1) molecular signature of hMSCs, associated with proinflammatory features, which is acquired following the coculture with the blood compartment. This suggests the establishment of an osteoblastic-like niche environment, mutually interacting with hematopoietic cells and capable of regulating HSPC activities, including proliferation and functional regulation.

The Engineered Niche Shares Structural and Compositional Features with Native Human BM.

The role of hMSCs in the establishment of a supportive environment for the hematopoietic compartment was further investigated by studying the organization and composition of the eN. Immunofluorescence analysis of thick construct sections revealed a homogeneous network formed by hMSCs within the scaffold (VENUS signal, Fig. 4A). This observed mesenchymal fraction consisted in 1.3 × 10⁶ hMSC-derived cells (Fig. 4B), as assessed by flow cytometry. The generated tissue resulting from the osteoblastic differentiation of hMSCs consisted in a dense human stroma filling the material pores and embedding mesenchymal cells (Fig. 4C). The composition of the deposited ECM included collagen type 1, collagen type 4, and fibronectin (Fig. 4C), reported as the main structural proteins of BM (38). Indeed, these were also abundantly found in healthy donor-derived BM specimens (human niche, Fig. 4D). The similarities between the human tissue and our engineered niche were not restricted to structural proteins. In human biopsies, osteocalcin staining revealed the presence of osteoblasts lining the bone surface (Fig. 4D). Remarkably, in the eN, a comparable pattern was observed by detection of osteocalcin in cells at the interface between the ceramic material and the cellular/ECM stroma (Fig. 4C).

Altogether, these results evidence the successful formation of a complex tissue under perfusion culture, associated with the development and support of human hematopoiesis (Fig. 2). The engineered environment was shown to share structural and compositional features typical of native human BM, suggesting the partial reconstitution of an osteoblastic-like niche environment.

The Bioreactor-Engineered Niche Displays a Functional Compartmentalization.

The developed culture system comprises two distinct phases: a liquid-fraction consisting in the supernatant (SN) SI Appendix, Fig. S6) and the stromal/ECM tissue confined within the scaffold chamber (ECM, SI Appendix, Fig. S6). This led us to further hypothesize that the two environments could differently impact the distribution of blood cell phenotypes. We thus separately analyzed HSPCs derived from these two phases by flow cytometry (SI Appendix, Fig. S6).

Despite the convection induced by perfusion flow, a specific cellular allocation was observed as 80% of the retrieved HSPC (CD34⁺/CD38⁻) populations resided in the ECM (Fig. 5A, Left). Conversely, more committed populations (CD34⁻/CD38⁺) exhibited a balanced distribution with 54% in ECM and 46% in SN (Fig. 5A, Right). A distinct pattern could be identified by further assessing the preferential localization of stem and progenitor populations, according to their progressive commitment (Fig. 5B). Remarkably, HSCs were found almost exclusively in the stroma (98%) (Fig. 5B) together with a vast majority of MPPs (76% in ECM) (Fig. 5B). This applied to a lower extent to common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs) (67% in ECM) while GMPs were more abundant in the SN (61%) (Fig. 5B). The localization was also shown to impact on the in vitro functionality of cells since CD34⁺ cells retrieved from ECM exhibited an increased capacity to form myeloid colonies compared with their SN counterpart (Fig. 5C). This includes the superior formation of GEmM, GM, and BFU colonies (2.6, 3.7, and 3.1 f.i., respectively).

The presence of human blood cells in the niche was confirmed by immunofluorescence, with human CD34⁺ cells abundantly detected within the ECM of the engineered tissue (Fig. 5D). Cells were predominantly located at the periphery of the scaffolds (Edge, Fig. 5D) but also largely infiltrated the tissue as shown by presence in the inner pores (Inner, Fig. 5D) and in the more central area (Central, Fig. 5D). Importantly, the mesenchymal and hematopoietic fractions in the engineered stroma were found within an organized ECM (Fig. 5E, Left) and closely interacted, as shown by explicit physical contacts (Fig. 5E, Right).

Altogether, this demonstrates the existence of a functional compartmentalization in the 3D perfusion system. The eN displays a hierarchical chemoattractant property on HSPC populations while commitment is progressively associated with a release into the liquid phase. The preferential stem cell confinement in the stroma, together with evidences of hMSC–CD34⁺ interactions, suggests an essential hMSC-triggered regulation of HSPC activities, resulting in a superior preservation of stem cell functionality.

BM Niches with Customized Molecular Signatures Can Be Genetically Engineered.

We next aimed at a proof of principle for the use of our system for the generation of customized hematopoietic niches with tailored molecular signature.

To this end, primary hMSCs were efficiently transduced using a VENUS-SDF1α lentivirus (>95%) (SI Appendix, Fig. S7 A and B), resulting in SDF1α overexpression (SI Appendix, Fig. S7C). SDF1α-engineered niches (eN-SDF1α) were subsequently produced, following the previously described protocol, and compared with eN without SDF1α enrichment. Throughout the culture period, substantial levels of SDF1α protein were continuously produced by the eN-SDF1α (>2,000 pg/mL/day/bioreactor) (Fig. 6A) compared with the eN (<300 pg/mL/day/bioreactor) (Fig. 6A).

When retrieved from the bioreactor chambers, the different engineered tissues were macroscopically identical (Fig. 6B). Immunofluorescence analysis revealed the presence of hMSCs in both niches, but eN-SDF1α displayed an increased presence of SDF1α protein, confirming the secretion pattern previously observed and validating the molecular customization of the niche. The protein was particularly abundant intracellularly within hMSCs directly lining on the ceramic surface (eN-SDF1α, Fig. 6B and SI Appendix, Fig. S7D).

Flow cytometry quantitative phenotypic analysis was performed to assess the effect of the SDF1α enrichment in the system. The total number of blood cells pooled from both compartments (ECM and SN) was similar in the eN and eN-SDF1α groups (SI Appendix, Fig. S8A). However, a marked reduction in the number of HSPCs was observed in the eN-SDF1α, reflected by a significant fold increase in HSCs (1.9 f.i.) and MPPs (1.4 f.i.) in the eN systems compared with the eN-SDF1α. Importantly, the SDF1α effect appeared to be restricted to these populations since the MLPs and general blood contents were identical in both niches (SI Appendix, Fig. S8A). Interestingly, observed differences were correlated with the system compartmentalization. Indeed, the SN cellular content of both eN and eN-SDF1α was identical in composition (SN, Fig. 6C). Instead, the ECM compartment of the eN showed a higher number of HSPCs, including superior content in HSCs and MPPs (2.1. and 1.6 f.i., respectively) while, again, the MLPs and global blood numbers were not affected.

We investigated the cell cycle status of HSPC, HSC, and MPP populations retrieved from our 3D conditions. This revealed a quiescence-inducing effect of SDF1α in eN-SDF1α, with a significant increase of HSPCs, MPPs, and HSCs in G0 phase (Fig. 6D and SI Appendix, Fig. S8 B and C). In particular, in cells retrieved from the ECM, we measured an increase of 29.6%, 36.1%, and 26.9% in the number of quiescent HSPCs, HSCs, and MPPs, respectively (Fig. 6D). Thus, the observed lower proportions of HSCs and MPPs in the ECM of eN-SDF1α niches were due to the quiescence induction by SDF1α overexpression.

These findings exemplify the possibility to use the described model to engineer customized BM niches, capable to specifically impact on HSC and MPP distribution and behavior in the system.

Perturbation of HSC Behavior by Simulation of Injury in Engineered Niche.

We further assessed whether the blood cell distribution can be perturbed by simulating a niche injury (SI Appendix, Fig. S9A). Before CD34⁺ loading, the eN were exposed to bleomycin (eN-Bleo), as potent drug inducing DNA damage (39). The drug altered hMSC metabolism (30% reduction of activity) (SI Appendix, Fig. S9B) but not their viability in the system (SI Appendix, Fig. S9C).

While both niches displayed similar HSPC content, the total number of HSCs was significantly higher in the eN-Bleo (SI Appendix, Fig. S9 D and E). The observed increase resulted from a higher HSC content in the ECM of eN-Bleo (Fig. 7A) while blood composition was identical in the SN of both eN and eN-Bleo (SI Appendix, Fig. S7A). Cells isolated from respective ECM niches showed a substantial decrease in the percentage of quiescent HSCs in eN-Bleo (8% vs. 20% in eN) (Fig. 7B). Interestingly, this was restricted to HSCs, and the cell cycle of HSPC and MPP populations was not affected.

These data indicate that the exposure to bleomycin impairs the capacity of hMSCs to maintain HSCs in a quiescent status, resulting in their increased proliferation. We thus validate the possibility to exploit our system for the study of human hematopoiesis in particular scenarios, like after injury.