Purification and functional characterization of novel human skeletal stem cell lineages - PubMed (original) (raw)

Review

. 2023 Jul;18(7):2256-2282.

doi: 10.1038/s41596-023-00836-5. Epub 2023 Jun 14.

Thomas H Ambrosi # 1 2 3, Holly M Steininger # 1, Lauren S Koepke # 1, Yuting Wang # 1 4, Liming Zhao 1 4, Matthew P Murphy 1 5, Alina A Alam 1, Elizabeth J Arouge 1, M Gohazrua K Butler 1, Eri Takematsu 1, Suzan P Stavitsky 1, Serena Hu 6, Debashis Sahoo 7, Rahul Sinha 1, Maurizio Morri 8 9, Norma Neff 8, Julius Bishop 6, Michael Gardner 6, Stuart Goodman 6, Michael Longaker 1 2 10, Charles K F Chan 11 12 13

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Review

Purification and functional characterization of novel human skeletal stem cell lineages

Malachia Y Hoover et al. Nat Protoc. 2023 Jul.

Abstract

Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.

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Figures

Extended Data Fig. 1 |

Extended Data Fig. 1 |. FACS gating strategy for other hSSC sources.

FACS gating plots for other three hSSC sources, including: fracture callus (28-year-old clavicle), femoral head (87-year-old) and iPSCs (human-monocyte-derived line, SCVI 113).PRO

Extended Data Fig. 2 |

Extended Data Fig. 2 |. FACS gating strategy for SSCs of bone marrow reamings.

FACS gating plots for SSCs collected from human bone marrow reamings of 22-year-old femur (top) and 87-year-old hip (bottom).

Extended Data Fig. 3 |

Extended Data Fig. 3 |. Fluorescence Minus One (FMO) gating strategy.

FMO controls help to define the negative signal for the given antibody and allow for a more informed gating strategy. Cells were isolated from a 76-year-old male femoral head. Representative plots were generated on a FACS Aria II for each FMO (eg. anti-Tie2/CD31 in APC-Cy7, anti-CD235/CD45/DAPI in Pacific Blue, anti-PDPN in APC, anti-CD146 in PE-Cy7, anti-CD73 in FitC, and anti-CD164 in PE).

Fig. 1 |

Fig. 1 |. Overview of the protocol for human skeletal stem cell isolation and functional assessment.

hSSCs can be isolated from multiple skeletal tissues and induced from pluripotent stem cells. The protocol includes isolation of the human skeletal elements by tissue dissection (Step 1A–D); dissociation of the skeleton into a single-cell suspension by mechanical (Step 1A–D) and enzymatic digestion (Steps 1A–D); depletion of red blood cells with ACK lysis buffer (Steps 2–3); antibody staining of the cells for skeletal lineage surface markers (Steps 4–14) and analysis and sorting of the cells on a flow cytometer (Steps 15–22); functional assessment of skeletal-lineage phenotype by in vivo xenograft (Box 1), renal subcapsular transplantation (Step 23A), in vitro colony-formation assay (Step 23B) and scRNAseq (Step 23E; UMAP, uniform manifold approximation and projection); analysis of grafts by micro CT, Movat Pentachrome and immunohistochemistry, and analysis of differentiation cultures by crystal violet (Step 23B), Alcian blue staining (Step 23C) or Alizarin red S staining (Step 23D).

Fig. 2 |

Fig. 2 |. hSSC sources, hierarchy and FACS gating strategy.

a, Representative sources of hSSCs: Fracture callus tissue of a 40-year-old male patient, femoral head of a 76-year-old patient, phalange bones of a 17-week-old fetus (gestational age) and iPSC-derived SSCs 2 weeks after induction (brightfield image). b, hSSC hierarchy and associated markers. The self-renewing hSSC gives rise to the multipotent hBCSP cells. The hBCSP may differentiate into either one of three chondroprogenitor populations (hCP1–3) responsible for cartilage formation, or the hOP responsible for bone and stroma formation. c, Representative FACS plots for human fetal bone sample. Variation between SSC and progenitor yields vary between sample tissue source and due to external factors, such as patient age, time since injury and patient health.

Fig. 3 |

Fig. 3 |. Subcutaneous human xenograft model and FACS isolation of hSSC lineages.

a, Phalange from a 17-week-old fetus is subcutaneously transplanted onto dorsum of a postnatal day-3 pup. Six weeks post-transplant, the fetal xenograft is subjected to micro CT, and then excised out of mouse for pentachrome staining. b, Representative FACS plots from three tissue components of the human fetal phalange xenograft: cartilage, bone and bone marrow. c, Gross images of human fetal phalanges pre- and post-implantation after 18 weeks. 1 = proximal phalanx, 2 = middle phalanx, 3 = distal phalanx. d, Quantification of length of proximal phalanx (PP) and digit (DP) shown in (c). (n = 3 specimens per group). Graphs show mean ± s.e.m. Two-tailed Student’s _t_-test. ****≤000005. e, Human nuclear staining (HNA) of fetal phalange subcutaneously transplanted. f, Xenografted fetal bone intramedullary injected with lentivirally GFP-labeled patient-derived hSSCs dissected in half. Inset: magnified image of GFP signal from lentivirally labeled hSSCs. Scale bars: 200 mm (a), 200 mm (e) and 1 mm (f).

Fig. 4 |

Fig. 4 |. Renal capsule xenograft model and in vitro skeletal differentiation assays.

a, Schematic of hSSC renal and intra-xenograft transplants. b, Step-by-step procedure of renal capsule transplant of hSSCs in NSG mice (details in Step 23A). c, Pentachrome staining of hSSC-derived grafts 4 weeks after transplantation beneath the renal capsule. d, Representative micro CT images of a patient-derived hSSC graft that was co-transplanted with Matrigel and BioOss (anorganic bovine cancellous bone; bottom) and a BioOss transplant without cells as control (top). e, In vitro assays: CFU-F test for clonogenicity of hSSCs stained by crystal violet, osteogenesis differentiation assay for bone formation potential stained by Alizarin red S and chondrogenesis differentiation assay for cartilage forming potential stained by Alcian blue. Scale bars: 1 mm (c) and 10 mm for CFU, 200 μm for osteogenesis and 5 mm for chondrogenesis (e).

Fig. 5 |

Fig. 5 |. ScRNAseq platform for analyzing hSSCs.

a, Step-by-step platform for processing hSSCs using SmartSeq2 and subsequent downstream analysis. b, Representative graphs of single-cell cDNA traces. c, Representative graph of pooled library trace using a Bioanalyzer. d, Example of quality filtering of 96 sequenced single cells of vertebral fracture hSSCs from a 40-year-old man for total RNA counts, number of genes expressed, percentage of mitochondrial genes and ribosomal genes. e, Representative UMAP clustering graph at resolution of 0.4, n_neighbors = 10 and n_pcs = 8 (based on principal component analysis elbow plot). f, Corresponding Leiden clustering graph. g, Representative heat map of top 30 genes for each cluster. h, Cluster marker genes that can be used for further separation and analysis of hSSC heterogeneity.

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