Adipocytic Cells Augment the Support of Primitive Hematopoietic Cells In Vitro But Have No Effect in the Bone Marrow Niche Under Homeostatic Conditions

AbstractIn bone marrow, hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor cells (HPCs) cooperate to differentiate and replenish blood and immune cells. It has long been recognized bone marrow niche parameters interact with hematopoietic stem and progenitor cells (HSPCs) and additional work is required to study niche physical signals controlling cell behavior. Here we presented that important biophysical signals, stiffness and dimensionality, regulating expansion of bone marrow HSPCs. Mice bone marrow derived progenitor cells were cultured in collagen I hydrogel in vitro. We found stiffer 3D matrix promoted the expansion of lineage negative (Lin−) progenitor cells and Lin−Sca-1+c-kit+ (LSK) HSPCs compared to softer hydrogel. Compared with cells cultured in 2D environment, 3D embedded construct had significant advantage on HSPCs expansion, accompanied by increases on myeloid and lymphoid lineage fractions. Bright changes on gene expression were subsequently discovered. According to these data, we concluded that culture matrix dimensionality is an important factor to regulate the behavior of subpopulations in hematopoietic cell pool, which should be considered in attempts to illuminate HSCs fate decision in vitro.Statement of SignificanceWe would like to submit the enclosed manuscript entitled "Importance of Niche-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool", which we wish to be considered for publication in Biophysical Journal. Studies about the interaction between HSCs and factors provided by their microenvironment is largely focus on pure perspective of biology. But biophysical factors affecting HSC fate and behavior needs to be further explore. Herein we found ex vivo culture dimensionality affected HSPC expansion. Cell surface marker detection and mRNA expression analysis predicted the changes on myeloid and lymphoid lineage fractions. We hope niche physical signals which we identified will be considered to design HSC biomimetic niches in clinical applications. And we believe that our study will make it interesting to general readers. We deeply appreciate your consideration of our manuscript, and we look forward to receiving comments from the reviewers.

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Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

Scientific Reports ◽

10.1038/s41598-021-85122-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marie-Theresa Weickert ◽

Judith S. Hecker ◽

Michèle C. Buck ◽

Christina Schreck ◽

Jennifer Rivière ◽

...

Keyword(s):

Bone Marrow ◽

Cell Fate ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Cell Types ◽

Bone Marrow Niche ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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Multimodal imaging of a 3D Bone Marrow Niche in vitro model to study dynamic response in pediatric leukemia

10.22443/rms.emc2020.977 ◽

2021 ◽

Author(s):

Giulia Borile ◽

Keyword(s):

Bone Marrow ◽

Dynamic Response ◽

Multimodal Imaging ◽

In Vitro Model ◽

Bone Marrow Niche ◽

Pediatric Leukemia ◽

Vitro Model

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Preclinical Evidence of Nanomedicine Formulation to Target Mycobacterium tuberculosis at Its Bone Marrow Niche

Pathogens ◽

10.3390/pathogens9050372 ◽

2020 ◽

Vol 9 (5) ◽

pp. 372 ◽

Cited By ~ 1

Author(s):

Jaishree Garhyan ◽

Surender Mohan ◽

Vinoth Rajendran ◽

Rakesh Bhatnagar

Keyword(s):

Bone Marrow ◽

Mycobacterium Tuberculosis ◽

In Vitro Release ◽

Bone Marrow Niche ◽

Mice Model ◽

Latently Infected ◽

Marrow Mesenchymal Stem Cells

One-third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). Recently, we found that dormant Mtb hides in bone marrow mesenchymal stem cells (BM-MSCs) post-chemotherapy in mice model and in clinical subjects. It is known that residual Mtb post-chemotherapy may be responsible for increased relapse rates. However, strategies for Mtb clearance post-chemotherapy are lacking. In this study, we engineered and formulated novel bone-homing PEGylated liposome nanoparticles (BTL-NPs) which actively targeted the bone microenvironment leading to Mtb clearance. Targeting of BM-resident Mtb was carried out through bone-homing liposomes tagged with alendronate (Ald). BTL characterization using TEM and DLS showed that the size of bone-homing isoniazid (INH) and rifampicin (RIF) BTLs were 100 ± 16.3 nm and 84 ± 18.4 nm, respectively, with the encapsulation efficiency of 69.5% ± 4.2% and 70.6% ± 4.7%. Further characterization of BTLs, displayed by sustained in vitro release patterns, increased in vivo tissue uptake and enhanced internalization of BTLs in RAW cells and CD271+BM-MSCs. The efficacy of isoniazid (INH)- and rifampicin (RIF)-loaded BTLs were shown using a mice model where the relapse rate of the tuberculosis was decreased significantly in targeted versus non-targeted groups. Our findings suggest that BTLs may play an important role in developing a clinical strategy for the clearance of dormant Mtb post-chemotherapy in BM cells.

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Pumpless Microfluidic System for Bone Marrow Niche-on-a-Chip in vitro Modelling and Multiphoton Imaging in Leukemia

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2574 ◽

2020 ◽

Vol 118 (3) ◽

pp. 463a

Author(s):

Giulia Borile ◽

Giulia Borella ◽

Camille Charoy ◽

Andrea Filippi ◽

Filippo Romanato ◽

...

Keyword(s):

Bone Marrow ◽

Microfluidic System ◽

Bone Marrow Niche ◽

Multiphoton Imaging ◽

In Vitro Modelling

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Preclinical Evaluation of a Bone-Marrow Autograft Culture Procedure for Generating Lymphokine-Activated Killer Cells in Vitro

Canadian Journal of Infectious Diseases ◽

10.1155/1992/234936 ◽

1992 ◽

Vol 3 (suppl b) ◽

pp. 123-127 ◽

Cited By ~ 3

Author(s):

Hans-Georg Klingemann ◽

Heather Deal ◽

Dianne Reid ◽

Connie J Eaves

Keyword(s):

Bone Marrow ◽

Calf Serum ◽

Cell Activity ◽

Hematopoietic Cells ◽

Lak Cells ◽

High Dose ◽

Lymphokine Activated Killer Cells ◽

Lak Cell

Despite the use of high dose chemoradiotherapy for the treatment of acute leukemia. relapse continues to be a major cause of death in patients given an autologous bone marrow transplant. Further augmentation of pretransplant chemotherapy causes life threatening toxicity to nonhematopoietic tissues and the effectiveness of currently available ex vivo purging methods in reducing the relapse rate is unclear. Recently, data from experimental models have suggested that bone marrow-derived lymphokine (IL-2)-activated killer (BM-LAK) cells might be used to eliminate residual leukemic cells both in vivo and in vitro. To evaluate this possibility clinically, a procedure was developed for culturing whole marrow harvests with IL-2 prior to use as autografts, and a number of variables examined that might affect either the generation of BM-LAK cells or the recovery of the primitive hematopoietic cells. The use of Dexter long term culture (LTC) conditions, which expose the cells to horse serum and hydrocortisone. supported LAK cell generation as effectively as fetal calf serum (FCS) -containing medium in seven-day cultures. Maintenance of BM-LAK cell activity after a further seven days of culture in the presence of IL-2 was also tested. As in the clinical setting. patients would receive IL-2 in vivo for an additional week immediately following infusion of the cultured marrow autograft. Generation ofBM-LAK activity was dependent on the presence of IL-2 and could be sustained by further incubation in medium containing IL-2. Primitive hematopoietic cells were quantitated by measuring the number of in vitro colony-forming progenitors produced after five weeks in secondary Dexter-type LTC. Maintenance of these 'LTC-initiating cells' was unaffected by lL-2 in the culture medium. These results suggest that LAK cells can be generated efficien tly in seven-day marrow autograft cultures containing IL-2 under conditions that allow the most primitive human hematopoietic cells currently detectable to be maintained.

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Primitive hematopoietic cells in murine bone marrow express the CD34 antigen

Blood ◽

10.1182/blood.v88.10.3774.bloodjournal88103774 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3774-3784 ◽

Cited By ~ 65

Author(s):

F Morel ◽

SJ Szilvassy ◽

M Travis ◽

B Chen ◽

A Galy

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Progenitor Cells ◽

Significant Proportion ◽

Hematopoietic Cells ◽

Full Detail ◽

Hematopoietic Stem ◽

Cd34 Antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.

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Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro

Transfusion Medicine and Hemotherapy ◽

10.1159/000520932 ◽

2022 ◽

pp. 1-10

Author(s):

Patrick Wuchter ◽

Anke Diehlmann ◽

Harald Klüter

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Stem Cell Niche ◽

Model Systems ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Hematopoietic Stem Cell Niche ◽

Cell Niche

Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

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