Purification and functional characterization of novel human skeletal stem cell lineages

Hoover, Malachia Y.; Ambrosi, Thomas H.; Steininger, Holly M.; Koepke, Lauren S.; Wang, Yuting; Zhao, Liming; Murphy, Matthew P.; Alam, Alina A.; Arouge, Elizabeth J.; Butler, M. Gohazrua K.; Takematsu, Eri; Stavitsky, Suzan P.; Hu, Serena; Sahoo, Debashis; Sinha, Rahul; Morri, Maurizio; Neff, Norma; Bishop, Julius; Gardner, Michael; Goodman, Stuart; Longaker, Michael; Chan, Charles K. F.

doi:10.1038/s41596-023-00836-5

Cited by 14 publications

(7 citation statements)

References 39 publications

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“…However, using bulk RNA-seq data cannot give detailed information about the heterogeneity of hBMSCs obtained from different isolation protocols. Therefore, future studies should take this into consideration for protocol validation as in vitro cultivation may prefer expanding specific cell types, which can be overlooked by functional assays without cell selection ( Hoover et al, 2023 ). Moreover, the molecular analyses of BMSC quality in vitro , it is also important to test stem cell potential using in vivo experiments depending on the context of the clinical studies ( Robey et al, 2014 ).…”

Section: Discussionmentioning

confidence: 99%

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Ferencakova,

Benova,

Raska

et al. 2023

Front. Cell Dev. Biol.

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Background: Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics.Methods: BM donors (age: 48–85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed.Results: The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs.Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.

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Section: Discussionmentioning

confidence: 99%

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Ferencakova,

Benova,

Raska

et al. 2023

Front. Cell Dev. Biol.

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“…Flow cytometry and cell sorting were performed on a FACS Aria II cell sorter (BD Biosciences) and analyzed using FlowJo software. Mouse long bones and human patient fracture callus samples (IRB-35711; SCRO-525) were dissected and freed from the surrounding soft tissue, which was then followed by dissociation with mechanical and enzymatic steps as described 45,46 . Briefly, the tissue was placed in collagenase digestion buffer supplemented with DNase and incubated at 37°C for 60 min under constant agitation.…”

Section: Methodsmentioning

confidence: 99%

“…Single ocSSCs from 4-week-old Esr1 fl/fl or Esr1 Nkx2-1Cre long bones were isolated via FACS using processing and flow cytometry protocol as described above. Single-cell suspension of four mice per group were pooled, and individual cells were captured in separate wells of a 96-well plate containing 4 µl lysis buffer (1 U/μl RNase inhibitor [Clontech, Cat#: 2313B]), 0.1% Triton (Thermo Fisher Scientific, Cat#: 85111), 2.5 mM dNTP (Invitrogen, Cat#: 10297–018), 2.5 μM oligo dT30VN (IDT, custom: 5′– AAGCAGTGGTATCAAC-GCAGAGTACT30VN -3′), and 1:600,000 External RNA Controls Consortium ExFold RNA Spike-In Mix 2 (ERCC; Invitrogen, Cat#: 4456739) in nuclease-free water (Thermo Fisher Scientific, Cat#: 10977023) according to a modified SmartSeq2 protocol 46 , 50 . Two 96-well plates per phenotype with a single ocSSCs per well were sorted and processed.…”

Section: Methodsmentioning

confidence: 99%

Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

Babey,

Krause,

Herber

et al. 2023

Preprint

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In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone resorption. While estrogen would normally counteract excessive bone loss and maintain sufficient bone formation during this postpartum period, this sex steroid drops precipitously after giving birth. Here, we report that brain-derived CCN3 (Cellular Communication Network factor 3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to promote bone mass in lactating females. Using parabiosis and bone transplant methods, we first established that a humoral factor accounts for the female-specific, high bone mass previously observed by our group after deleting estrogen receptor alpha (ERα) from ARCKISS1neurons1. This exceptional bone phenotype in mutant females can be traced back to skeletal stem cells (SSCs), as reflected by their increased frequency and osteochondrogenic potential. Based on multiple assays, CCN3 emerged as the most promising secreted pro-osteogenic factor from ARCKISS1neurons, acting on mouse and human SSCs at low subnanomolar concentrations independent of age or sex. That brain-derived CCN3 promotes bone formation was further confirmed by in vivo gain- and loss-of-function studies. Notably, a transient rise in CCN3 appears in ARCKISS1neurons in estrogen-depleted lactating females coincident with increased bone remodeling and high calcium demand. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone that defines a novel female-specific brain-bone axis for ensuring mammalian species survival.

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“…This approach enables the identification of cell types, states, functions, and relationships, thus offering an unprecedented opportunity to uncover subpopulations with shared gene expression profiles within heterogeneous cell populations. Although scRNA-seq has revealed the heterogeneity of BMSCs and Wharton's Jelly-derived MSCs, and identified subpopulations with unique functions 15 , 16 , the presence of subpopulations related to neural regeneration within MSCs remains largely unknown.…”

Section: Introductionmentioning

confidence: 99%

Exosomes derived from CD271⁺CD56⁺ bone marrow mesenchymal stem cell subpopoulation identified by single-cell RNA sequencing promote axon regeneration after spinal cord injury

Sun,

Liu,

Qin

et al. 2024

Theranostics

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Rationale: Spinal cord injury (SCI) results in neural tissue damage. However, the limited regenerative capacity of adult mammals' axons upon SCI leads to persistent neurological dysfunction. Thus, exploring the pathways that can enhance axon regeneration in injured spinal cord is of great significance. Methods: Through the utilization of single-cell RNA sequencing in this research, a distinct subpopulation of bone marrow mesenchymal stem cells (BMSCs) that exhibits the capacity to facilitate axon regeneration has been discovered. Subsequently, the CD271 + CD56 + BMSCs subpopulation was isolated using flow cytometry, and the exosomes derived from this subpopulation (CD271 + CD56 + BMSC-Exos) were extracted and incorporated into a hydrogel to create a sustained release system. The aim was to investigate the therapeutic effects of CD271 + CD56 + BMSC-Exos and elucidate the underlying mechanisms involved in promoting axon regeneration and neural function recovery. Results:The findings indicate that CD271 + CD56 + BMSC-Exos share similar physical and chemical properties with conventional exosomes. Importantly, in an SCI model, in situ implantation of CD271 + CD56 + BMSC-Exos hydrogel resulted in increased expression of NF and synaptophysin, markers associated with axon regeneration and synapse formation, respectively. This intervention also contributed to improved neural function recovery.In vitro experiments demonstrated that CD271 + CD56 + BMSC-Exos treatment significantly enhanced axon extension distance and increased the number of branches in dorsal root ganglion axons. Moreover, further investigation into the molecular mechanisms underlying CD271 + CD56 + BMSC-Exos-mediated axon regeneration revealed the crucial involvement of the miR-431-3p/RGMA axis. Conclusion: In summary, the implantation of CD271 + CD56 + BMSC-Exos hydrogel presents a promising and effective therapeutic approach for SCI.

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Purification and functional characterization of novel human skeletal stem cell lineages

Cited by 14 publications

References 39 publications

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

Exosomes derived from CD271⁺CD56⁺ bone marrow mesenchymal stem cell subpopoulation identified by single-cell RNA sequencing promote axon regeneration after spinal cord injury

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Purification and functional characterization of novel human skeletal stem cell lineages

Cited by 14 publications

References 39 publications

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

Exosomes derived from CD271+CD56+ bone marrow mesenchymal stem cell subpopoulation identified by single-cell RNA sequencing promote axon regeneration after spinal cord injury

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Product

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Exosomes derived from CD271⁺CD56⁺ bone marrow mesenchymal stem cell subpopoulation identified by single-cell RNA sequencing promote axon regeneration after spinal cord injury