Prospective isolation of defined cell types is critical for the functional study of stem cells, especially in primary human tissues. Here, we present a protocol for purifying 10 transcriptomically and functionally distinct neural stem and progenitor cell types from the developing human brain using fluorescence-activated cell sorting. We describe steps for tissue dissociation, staining, and cell sorting as well as downstream functional experiments for measuring clonogenicity, differentiation, and engraftment potential of purified populations. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

Mammalian erythropoiesis involves progressive chromatin compaction and subsequent enucleation in terminal differentiation, but the mechanisms underlying the three-dimensional chromatin reorganization remain obscure. Here, we systematically analyze the higher-order chromatin in purified populations of primary human erythroblasts. Our results reveal that heterochromatin regions undergo substantial compression, with H3K9me3 markers relocalizing to the nuclear periphery and forming a significant number of long-range interactions, and that ~58% of the topologically associating domain (TAD) boundaries are disrupted, while certain TADs enriched for markers of the active transcription state and erythroid master regulators, GATA1 and KLF1, are selectively maintained during terminal erythropoiesis. Finally, we demonstrate that GATA1 is involved in safeguarding selected essential chromatin domains during terminal erythropoiesis. Our study therefore delineates the molecular characteristics of a development-driven chromatin compaction process, which reveals transcription competence as a key indicator of the selected domain maintenance to ensure appropriate gene expression during the extreme compaction of chromatin.
© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

Development of mammalian red blood cells involves progressive chromatin compaction and subsequent enucleation in terminal stages of differentiation, but the molecular mechanisms underlying the three-dimensional chromatin reorganization and compaction remains obscure. Here, we systematically analyze the distinct features of higher-order chromatin in purified populations of primary human erythroblasts. Our results reveal that while heterochromatin regions undergo substantial compression, select transcription competent regions with active transcription signature are preferentially maintained to achieve a highly-compacted yet functional chromatin state in terminal erythropoiesis, which is about 20-30% of the nuclear volume compared to that of erythroid progenitors. While the partition of euchromatic and heterochromatic regions (compartment A and B) remain mostly unchanged, H3K9me3 marks relocalize to the nuclear periphery and a significant number of H3K9me3 long-range interactions are formed in the three-dimensional rewiring during terminal erythroid chromatin condensation. Moreover, ∼63% of the topologically associating domain (TAD) boundaries are disrupted, while certain TADs with active chromatin modification are selectively maintained during terminal erythropoiesis. The most well-maintained TADs are enriched for chromatin structural factors CTCF and SMC3, as well as factors and marks of the active transcription state. Finally, we demonstrate that the erythroid master regulator GATA1 involves in safeguarding select essential chromatin domains during terminal erythropoiesis. Our study therefore delineate the molecular characteristics of a development-driven chromatin compaction process, which reveals transcription competence as a key determinant of the select domain maintenance to ensure appropriate gene expression during immense chromatin compaction.