X41 - Developing 3D Vascularized Bone Marrow Organoids for B Cell Differentiation

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

IJ

Ishita Jain

Postdoctoral Scientist
Georgia Institute of Technology
Mountain View, California, United States

Co-Author(s)

CC

Christopher Carlson

Graduate Student
Georgia Institute of Technology, United States
AG

Andres J. Garcia, PhD

Professor
Georgia Institute of Technology, United States

Last Author(s)

AS

Ankur Singh

Professor
Georgia Institute of Technology, United States

Primary Investigator(s)

AS

Ankur Singh

Professor
Georgia Institute of Technology, United States

Introduction: : The bone marrow (BM) microenvironment is critical for hematopoietic stem and progenitor cell (HSPC) maintenance and differentiation, particularly for B cell lineage specification. Conventional 2D cultures lack the spatial and cellular complexity of the native BM niche, limiting their ability to fully recapitulate B cell developmental cues. To address this, we developed a 3D PEG-based hydrogel system engineered to mimic the BM microenvironment by incorporating key stromal and vascular components, aiming to support robust B lymphopoiesis in vitro.

Materials and

Methods: : The bone marrow organoids were prepared using PEG-Maleimide hydrogel functionalized with matrix-binding (RGD and REDV) and proteolytically degradable peptides (VPM) to permit cell adhesion and remodeling. Human osteoblasts differentiated from MSCs (mesenchymal stem cells) were encapsulated to mimic the endosteal niche, while primary CD34+ HSPCs were seeded to initiate hematopoiesis. To introduce vascular features, human umbilical vein endothelial cells (HUVECs) were incorporated around the bone marrow organoids to facilitate vascularization. B cell differentiation culture conditions were optimized for 32 days. Tissues were analyzed using flow cytometry and immunofluorescence to assess B cell markers and endothelial integration.

Results, Conclusions, and Discussions:: The PEG hydrogel-based organoids supported long-term survival and differentiation of CD34+ HSPCs with consistent different towards B cells lineage. At early timepoints, specifically Day 4, we observed the highest early B cell progenitor population (CD34+, CD45+, and CD19 low) that decreases at Day 8. Consequently, a significant increase in Pro B cell population (CD34+, CD45+, CD19 high and CD38+) is observed at Day 8. The inclusion of osteoblasts in the PEG hydrogels demonstrated greater than 2-fold increase in both the early B cell progenitor and pro B cell population. Furthermore, incorporation of HUVECs led to self-assembly of perfusable vascular-like networks within the hydrogel, confirmed by CD31+ network like structures.

Our findings demonstrate that a modular PEG hydrogel scaffold can effectively support 3D BM organoid formation with integrated vascular and osteogenic niches. We observed structural attributes of vascularization by HUVECs and are currently evaluating the effect of vascularization on enhanced B cell development. functionally enhanced B cell differentiation. Furthermore, we plan to study spatial colocalization of endothelial and hematopoietic cells as observed in in vivo marrow architecture. This model provides a valuable platform for studying human hematopoiesis and lymphopoiesis in a tunable, physiologically relevant context, with potential applications in disease modeling, immunotoxicity testing, and regenerative medicine.

Acknowledgements and/or References (Optional): :