A 3D Niche Nanobioreactor Supports Long-Term Tracking of Hematopoietic Stem and Progenitor Cell Cycle Kinetics, Self-Renewal and Cell Fate Determination

Introduction

Maintenance of hematopoietic stem cell (HSC) fitness is predicated on tightly regulated cell cycle transit, self-renewal and multi-lineage differentiation potential in supportive niches but can be altered detrimentally by inflammatory microenvironments and difficult to quantify. Here, we report on the development of a NASA-funded 3D biosensing nanobioreactor system (Pham...Jamieson. BioRxiv 2024) that enables real-time confocal fluorescent microscopic tracking of HSPC cell cycle kinetics with our dual fluorescence lentiviral FUCCI2BL cell cycle indicator (Pineda...Jamieson. Scientific Reports 2016), quantification of ADAR1 self-renewal gene activity with a lentiviral ADAR1 GFP reporter and cell fate decisions in response to niche cues over a 4 to 6 week period.

Methods

Niche nanobioreactors were constructed from 2-port, fluoroethyl polymer (FEP) film bags that are gas permeable, chemically inert, and transparent for imaging. A porcine gelatin sponge (J&J MedTech) matrix was cut to size to function as a physical scaffold before resealing with a heat sealer under sterile conditions. Subsequently, these nanobioreactors were utilized in experiments involving aged bone marrow samples collected from routine knee and hip replacement surgeries under an IRB-approved protocol. Upon collection, mononuclear cells were isolated by Ficoll-Paque density centrifugation and subjected to CD34+ immunomagnetic bead selection. The CD34^- bone marrow stromal cells were either seeded directly into the nanobioreactor following magnetic bead selection, or irradiated at 80 cGy prior to seeding into the nanobioreactor. The CD34⁺ cells were lentivirally transduced with the FUCCI2BL or our ADAR1-NanoLuc-GFP reporter (Crews...Jamieson. Cell Stem Cell 2023) and cultured for 48 hours or directly seeded into the same nanobioreactor as the autologous CD34^- cell fraction in a 1:4 ratio. At baseline, cells were stained for immune cell lineage markers CD3, CD19, CD14, and CD56, as well as a panel of additional surface markers and evaluated by FACS analysis to assess the frequency of progenitor cell subsets. The nanobioreactor cultures were analyzed weekly for up to 6 weeks. Cultures were also analyzed at baseline and weekly by whole transcriptome RNA sequencing (RNA-seq) and single cell RNA sequencing (sc-RNA-seq).

Results

Both RNA-seq and weekly FACS analysis demonstrated that a population of ADAR1-expressing hematopoietic stem cells (HSCs, CD34⁺CD38^-Lin^-) can be consistently detected in the nanobioreactor for four to six weeks as can FUCCI-red dormant HSCs. Upon four weeks of culture, we also observed the maintenance of progenitor populations such as common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), granulocyte-macrophage progenitors (GMP), multipotent progenitors (MPP), and lymphoid-primed multipotent progenitors (LMPP). When compared to traditional 2D cell culture, the 3D nanobioreactor system performs significantly better in maintaining populations of stem and progenitor cells. Furthermore, unirradiated CD34- cell populations, including immune cells, are also maintained in the nanobioreactor. Over a four week period, a stable population of T cells is detected, while B cells persist at low frequencies. Additionally, myeloid cells are maintained and, in some cases, expanded in the nanobioreactor.

Conclusions

Our 3D biosensing nanobioreactor culture system enables the prolonged maintenance of primary human HSPCs and effectively supports the culture of immune cells. This system can serve as an in vitro model system to study both HSPC and niche dynamics. Further investigation into additional immune cell subsets and their functionality following culture in the nanobioreactor is currently underway.