Haematopoietic development and HSC formation in vitro: promise and limitations of gastruloid models

Abstract

Haematopoietic stem cells (HSCs) are the most extensively studied adult stem cells. Yet, six decades after their first description, reproducible and translatable generation of HSC in vitro remains an unmet challenge. HSC production in vitro is confounded by the multi-stage nature of blood production during development. Specification of HSC is a late event in embryonic blood production and depends on physical and chemical cues which remain incompletely characterised. The precise molecular composition of the HSC themselves is incompletely understood, limiting approaches to track their origin in situ in the appropriate cellular, chemical and mechanical context. Embryonic material at the point of HSC emergence is limiting, highlighting the need for an in vitro model of embryonic haematopoietic development in which current knowledge gaps can be addressed and exploited to enable HSC production. Gastruloids are pluripotent stem cell-derived 3-dimensional (3D) cellular aggregates which recapitulate developmental events in gastrulation and early organogenesis with spatial and temporal precision. Gastruloids self-organise multi-tissue structures upon minimal and controlled external cues, and are amenable to live imaging, screening, scaling and physicochemical manipulation to understand and translate tissue formation. In this review, we consider the haematopoietic potential of gastruloids and review early strategies to enhance blood progenitor and HSC production. We highlight possible strategies to achieve HSC production from gastruloids, and discuss the potential of gastruloid systems in illuminating current knowledge gaps in HSC specification.

Keywords: gastruloid models; haematopoietic stem cells; pluripotent stem cells.

Figure 1.. Schematic diagram of approaches to generate HSCs.

Methods able to generate de novo transplantable hematopoietic stem and progenitor cells (HSPC) from ESCs/PSCs remain rare. Most approaches rely on mimicking HSC-niche interactions (via teratoma or embryoid body (EB) induction) and/or overexpression of transcription factors (TFs). Protocols that do not achieve engraftment (e.g. OP9-co-culture approaches by themselves do not support long-term transplantation potential) and those that do not use embryonic/pluripotent material as a starting point (e.g. the use of CD34+ cells isolated from peripheral blood) (envisioned in the left part of the figure) are out of the scope of our review and are noted here for completion only. Image is created with Biorender.com.

Figure 2.. Schematic representation of current protocols that mimic developmental hematopoiesis.

Black timeline represents the starting point of each protocol, up until the termination. (A) Protocol used by Rossi et al. [76], where 300–700 cells were aggregated into 96 well-round bottom plates cultured in N2B27. A pulse of CHI99021 was added between 48 and 72 h. Post-96 h, the aggregates were transferred into 24-well plates shaking on orbital shaker (as shown in grey box) and cultured in N2B27, VEGF, bFGF and AA until 14 4h. After 144 h the aggregates are cultured in N2B27 alone. Flow cytometry parameters are illustrated in black with the emergence of each marker corresponding to the timeline. (B) Protocol by Ragusa et al. [77], where 250 cells were aggregated in N2B27 into 96 well-round bottom ultra-low adherent plates as shown in grey box. A pulse of CHI99021 + Activin A is added between 48 and 72 h. Gastruloids are then cultured in N2B27, VEGF and FGF2. Shh was added between 144 and 168 h. Other growth factor cytokines (SCF, thrombopoietin — TPO, and Flt3L) are added in the last 2 days along with just N2B27 and VEGF. Flow cytometry parameters are illustrated in black with the emergence of each marker corresponding to the timeline.

Figure 3.. Summary of biological and experimental considerations for successful haemogenic gastruloid protocols.

Production of haematopoietic stem cells (HSC) from in vitro haemogenic gastruloid protocols rely on the recapitulation of key biological events in haematopoiesis. Different types of in vitro adaptations can improve current protocols by tackling mechanical, metabolic and epigenetic characteristics of physiological processes.