We are developing an artificial bone marrow for ex vivo culture of hematopoietic stem cells (HSCs). This platform has significant scientific value for testing hypotheses regarding the cascade of external signals responsible for directing HSC fate decisions within the bone marrow. An artificial marrow would also have significant clinical value for therapeutic expansion of HSCs or for study of the etiology and treatment of hematopoietic pathologies. However complicating this effort is limited information regarding the regulatory role played by the continuum of sub-niches that exist in close spatial order across the marrow responsible for maintaining hematopoietic homeostasis. We have recently described microfabrication approaches to generate an engineered bone marrow (EBM) containing overlapping patterns of marrow-inspired cellular, biophysical, and biomolecular cues to begin to examine the coordinated impact of these signals on HSC quiescence vs. activation. However, the small scale that makes the EBM advantageous introduces concerns regarding the unknown heterogeneity of a stem cell's response to these cues. While not surprising HSCs may exhibit a range of responses to a niche signal, effects may be magnified in multi-cue environments. We therefore propose to demonstrate a label-free approach to temporally track and quantify the heterogeneity of single HSC fate decisions in response to multiplexed EBM niche signals. To do this, we will combine photonic crystal enhanced microscopy (PCEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to create an integrated detection instrument able to trace single HSC fate decisions. We hypothesize that individually profiling these decisions across a continuum of biomaterial sub-niches will allow us to better predict niche regulation of HSC fate than more traditional metrics that report ensemble averages.
Aim 1 will integrate SIMS and PCEM to determine the heterogeneity of HSC response to biophysical cues demonstrated to have an effect on populations of HSCs.
Aim 2 will employ the PCEM/SIMS-based biosensor to examine HSC fate decisions within multi-cell colonies containing HSCs and supportive niche cells. Combining this novel biosensor with traditional functional assays will allow us, for the firt time, to determine the heterogeneity of HSC response to engineered niche signals. It also offers a framework to rapidly assess the impact of multiple signals using a minimal number of cells in order to identify hierarchies and/or synergies between these cues. Such data will bring new richness to our understanding of how HSCs integrate niche signals as well as identify critical design elements of an engineered bone marrow. By quantifying the level of heterogeneity in these fate decisions, we will also establish where along the continuum between single HSC and ensemble averages future investigations must focus.
An artificial bone marrow has significant clinical value for therapeutic expansion of hematopoietic stem cells as well as study of the etiology and treatment of hematopoietic diseases. While the hematopoietic system offers unique advantages for developing broadly applicable tools to dissect how external signals shape cell fate, poor understanding of the heterogeneity of a stem cell's response to a given cue remains a fundamental limit to progress. Here we combine orthogonal mass spectrometry and photonic crystal analysis techniques to create a label-free detection instrument to map the heterogeneity of HSC response to marrow-inspired cell and matrix signals.
