Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy

Abstract

A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely-related short-term repopulating HSCs (ST-HSCs), and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four sub-populations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition, and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that elicited them.

Combining Multiplex SERS Nanovectors and Multivariate Analysis for In Situ Profiling of Circulating Tumor Cell Phenotype Using a Microfluidic Chip

“…We further explore classification based on the abundant spectroscopic data from single cells. PLS regression model combined with linear discriminate analysis is a powerful statistical method for classifying and discriminating subtle variations in a dataset . Employing the fundamental principle of principle component analysis, PLS‐DA not only lowers the dimensions of spectral data from hundreds of wavenumbers to a few key components, but further rotate the components to separate the groups maximally.…”

Marrow-inspired matrix cues rapidly affect early fate decisions of hematopoietic stem and progenitor cells

“…Development of new tools to trace single HSC fate decisions with minimal sample manipulation and increased sensitivity is highly desirable to better resolve nuances of HSC-matrix interactions. Recent efforts in the area of time-lapse HSC tracing ( 14 ), Raman spectroscopy–based single HSC classification tools ( 50 ), and label-free photonic crystal imaging tools that can trace cell-matrix engagement ( 51 ) offer exciting opportunities for functionally phenotyping HSC fate specification events across a range of engineered bone marrow microenvironments. The observed gradations in matrix ligand composition and mechanical properties across the bone marrow may be directly linked to the differential regulation of HSC fate and motivate ongoing approaches using graded material platforms to support hematopoietic homeostasis ex vivo ( 52 , 53 ).…”

Mesenchymal stromal cell remodeling of a gelatin hydrogel microenvironment defines an artificial hematopoietic stem cell niche

“…The LSK faction was collect in PBS + 25% FBS on ice and immediately used [19,29,46,[67][68][69][70].…”