Label-free enrichment of primary human skeletal progenitor cells using deterministic lateral displacement

Abstract We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FACS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained within the CD34+ cell fraction. In the present study, the frequency of stromal progenitors in both the CD34+ and CD34− subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34+, CD38−, HLA-DR−, CDw90+ phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34−, CD38−, HLA-DR−, CDw90+ phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34+ fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34+ or CD34− bone marrow cells using reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34+ or CD34− cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34+ bone marrow cells, although the differences between CD34+ and CD34− stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/mL SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment.

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Label-free mesenchymal stem cell enrichment from bone marrow samples by inertial microfluidics

Analytical Methods ◽

10.1039/c7ay02500a ◽

2018 ◽

Vol 10 (7) ◽

pp. 713-721 ◽

Cited By ~ 13

Author(s):

Lap Man Lee ◽

Jenna M. Rosano ◽

Yi Wang ◽

George J. Klarmann ◽

Charles J. Garson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Bone Marrow Aspirate ◽

Label Free ◽

Therapeutic Implications ◽

Cartilage Reconstruction ◽

Cell Enrichment ◽

And Cartilage

Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical need in regenerative medicine such as orthopedic and cartilage reconstruction with important clinical and therapeutic implications.

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Faculty Opinions recommendation of Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.11493958.12528059 ◽

2011 ◽

Author(s):

Devendra Agrawal ◽

Vineet Agrawal

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Progenitor Cells ◽

Stem Cell Niche ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cell Niche

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Label-Free Rapid Separation and Enrichment of Bone Marrow-Derived Mesenchymal Stem Cells from a Heterogeneous Cell Mixture Using a Dielectrophoresis Device

Sensors ◽

10.3390/s18093007 ◽

2018 ◽

Vol 18 (9) ◽

pp. 3007 ◽

Cited By ~ 5

Author(s):

Junya Yoshioka ◽

Yu Ohsugi ◽

Toru Yoshitomi ◽

Tomoyuki Yasukawa ◽

Naoki Sasaki ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Gradient Method ◽

Label Free ◽

Rapid Separation ◽

Isolation System ◽

Tissue Samples ◽

Cell Based Therapy

Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes. Upon application of an electric field, HL-60 cells as models of promyelocytes aggregated and floated between the saw-shaped electrodes, while UE7T-13 cells as models of BMSCs were effectively captured on the tips of the saw-shaped electrodes. After washing out the HL-60 cells from the device selectively, the purity of the UE7T-13 cells was increased from 33% to 83.5% within 5 min. Although further experiments and optimization are required, these results show the potential of the DEP device as a label-free rapid cell isolation system yielding high purity for rare and precious cells such as BMSCs.

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The Use of Endothelial Progenitor Cells for the Regeneration of Musculoskeletal and Neural Tissues

Stem Cells International ◽

10.1155/2017/1960804 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Naosuke Kamei ◽

Kivanc Atesok ◽

Mitsuo Ochi

Keyword(s):

Bone Marrow ◽

Clinical Trials ◽

Endothelial Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Tissue Repair ◽

Cell Populations ◽

Neural Tissues ◽

Cell Source ◽

Stem Cell Populations

Endothelial progenitor cells (EPCs) derived from bone marrow and blood can differentiate into endothelial cells and promote neovascularization. In addition, EPCs are a promising cell source for the repair of various types of vascularized tissues and have been used in animal experiments and clinical trials for tissue repair. In this review, we focused on the kinetics of endogenous EPCs during tissue repair and the application of EPCs or stem cell populations containing EPCs for tissue regeneration in musculoskeletal and neural tissues including the bone, skeletal muscle, ligaments, spinal cord, and peripheral nerves. EPCs can be mobilized from bone marrow and recruited to injured tissue to contribute to neovascularization and tissue repair. In addition, EPCs or stem cell populations containing EPCs promote neovascularization and tissue repair through their differentiation to endothelial cells or tissue-specific cells, the upregulation of growth factors, and the induction and activation of endogenous stem cells. Human peripheral blood CD34(+) cells containing EPCs have been used in clinical trials of bone repair. Thus, EPCs are a promising cell source for the treatment of musculoskeletal and neural tissue injury.

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The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽

10.1182/blood.2021013954 ◽

2021 ◽

Author(s):

Yuqing Yang ◽

Andrew J Kueh ◽

Zoe Grant ◽

Waruni Abeysekera ◽

Alexandra L Garnham ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Bone Marrow Cells ◽

High Rate ◽

Embryonic Patterning ◽

Self Renewal ◽

Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

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Heparanase regulates retention and proliferation of primitive Sca-1+/c-Kit+/Lin− cells via modulation of the bone marrow microenvironment

Blood ◽

10.1182/blood-2007-10-116145 ◽

2008 ◽

Vol 111 (10) ◽

pp. 4934-4943 ◽

Cited By ~ 27

Author(s):

Asaf Spiegel ◽

Eyal Zcharia ◽

Yaron Vagima ◽

Tomer Itkin ◽

Alexander Kalinkovich ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Cathepsin K ◽

Hematopoietic Progenitors ◽

Hematopoietic Progenitor ◽

Cell Mobilization ◽

Tumor Growth And Metastasis

Abstract Heparanase is involved in tumor growth and metastasis. Because of its unique cleavage of heparan sulfate, which binds cytokines, chemokines and proteases, we hypothesized that heparanase is also involved in regulation of early stages of hematopoiesis. We report reduced numbers of maturing leukocytes but elevated levels of undifferentiated Sca-1+/c-Kit+/Lin− cells in the bone marrow (BM) of mice overexpressing heparanase (hpa-Tg). This resulted from increased proliferation and retention of the primitive cells in the BM microenvironment, manifested in increased SDF-1 turnover. Furthermore, heparanase overexpression in mice was accompanied by reduced protease activity of MMP-9, elastase, and cathepsin K, which regulate stem and progenitor cell mobilization. Moreover, increased retention of the progenitor cells also resulted from up-regulated levels of stem cell factor (SCF) in the BM, in particular in the stem cell–rich endosteum and endothelial regions. Increased SCF-induced adhesion of primitive Sca-1+/c-Kit+/Lin− cells to osteoblasts was also the result of elevation of the receptor c-Kit. Regulation of these phenomena is mediated by hyperphosphorylation of c-Myc in hematopoietic progenitors of hpa-Tg mice or after exogenous heparanase addition to wildtype BM cells in vitro. Altogether, our data suggest that heparanase modification of the BM microenvironment regulates the retention and proliferation of hematopoietic progenitor cells.

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Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells

Blood ◽

10.1182/blood.v87.10.4100.bloodjournal87104100 ◽

1996 ◽

Vol 87 (10) ◽

pp. 4100-4108 ◽

Cited By ~ 61

Author(s):

N Okumura ◽

K Tsuji ◽

Y Ebihara ◽

I Tanaka ◽

N Sawai ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Stromal Cells ◽

Stem Cell Factor ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Murine Bone Marrow ◽

Checkerboard Assay

We investigated the effects of stem cell factor (SCF) on the migration of murine bone marrow hematopoietic progenitor cells (HPC) in vitro using a modification of the checkerboard assay. Chemotactic and chemokinetic activities of SCF on HPC were evaluated by the numbers of HPC migrated on positive and negative gradients of SCF, respectively. On both positive and negative gradients of SCF, HPC began to migrate after 4 hours incubation, and their numbers then increased time- dependently. These results indicated that SCF functions as a chemotactic and chemokinetic agent for HPC. Analysis of types of colonies derived from the migrated HPC showed that SCF had chemotactic and chemokinetic effects on all types of HPC. When migrating activities of other cytokines were examined, interleukin (IL)-3 and IL-11 also affected the migration of HPC, but the degrees of each effect were lower than that of SCF. The results of the present study demonstrated that SCF is one of the most potent chemotactic and chemokinetic factors for HPC and suggest that SCF may play an important role in the flow of HPC into bone marrow where stromal cells constitutively produce SCF.

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Combining Electrostatic, Hindrance and Diffusive Effects for Predicting Particle Transport and Separation Efficiency in Deterministic Lateral Displacement Microfluidic Devices

Biosensors ◽

10.3390/bios10090126 ◽

2020 ◽

Vol 10 (9) ◽

pp. 126

Author(s):

Valentina Biagioni ◽

Giulia Balestrieri ◽

Alessandra Adrover ◽

Stefano Cerbelli

Keyword(s):

Particle Transport ◽

Separation Efficiency ◽

Lateral Displacement ◽

Operating Conditions ◽

Separation Performance ◽

Label Free ◽

Deterministic Lateral Displacement ◽

Promising Tool ◽

Label Free Detection ◽

Diffusive Effects

Microfluidic separators based on Deterministic Lateral Displacement (DLD) constitute a promising technique for the label-free detection and separation of mesoscopic objects of biological interest, ranging from cells to exosomes. Owing to the simultaneous presence of different forces contributing to particle motion, a feasible theoretical approach for interpreting and anticipating the performance of DLD devices is yet to be developed. By combining the results of a recent study on electrostatic effects in DLD devices with an advection–diffusion model previously developed by our group, we here propose a fully predictive approach (i.e., ideally devoid of adjustable parameters) that includes the main physically relevant effects governing particle transport on the one hand, and that is amenable to numerical treatment at affordable computational expenses on the other. The approach proposed, based on ensemble statistics of stochastic particle trajectories, is validated by comparing/contrasting model predictions to available experimental data encompassing different particle dimensions. The comparison suggests that at low/moderate values of the flowrate the approach can yield an accurate prediction of the separation performance, thus making it a promising tool for designing device geometries and operating conditions in nanoscale applications of the DLD technique.

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The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

Blood ◽

10.1182/blood.v85.9.2422.bloodjournal8592422 ◽

1995 ◽

Vol 85 (9) ◽

pp. 2422-2435 ◽

Cited By ~ 107

Author(s):

EK Waller ◽

J Olweus ◽

F Lund-Johansen ◽

S Huang ◽

M Nguyen ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Progenitor Cells ◽

Cell Sorting ◽

Single Cells ◽

Light Scatter ◽

Hematopoietic Progenitor ◽

Individual Culture ◽

Hla Dr ◽

Growth Properties

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

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