Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Uslu, Merve; Albayrak, Esra; Kocabaş, Fatih

doi:10.1002/jcp.29777

Cited by 25 publications

(20 citation statements)

References 92 publications

(143 reference statements)

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“…Supernatants which contain desired cells, were collected from the ow tube without removing inside BD IMagnet. The isolated cells were seeded on 96 well-plate in expansion medium at a density of 30,000 cells per well for ow cytometry analysis as we have done previously (Uslu et al, 2020).…”

Section: Lineage Negative Cell Isolationmentioning

confidence: 99%

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Pluripotin facilitates the expansion of hematopoietic stem cells, but restricts the growth of fibroblasts and the proliferation of mesenchymal stem cells from the bone marrow

Turan

Kocabaş

2021

Preprint

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Hematopoietic stem/progenitor cell (HSPC) quiescence modulators play a key role in the modulation of HSPC numbers. Targeting HSPC modulators with small molecules could contribute to the cell cycle entry and expansion of HSPC. To this end, we have investigated the effect of two small molecules Pluripotin and CHIR-99021 on HSPC expansion. Both Pluripotin and CHIR-99021 have been shown to result in a 3-fold rise in the murine pool of HSPC in a dose-dependent compartment after 7 days of treatments. In addition, we investigated the influence of Pluripotin treatment on the ex vivo expansion of human umbilical cord blood and bone marrow mononuclear cells. In specific, Pluripotin treatment increased human CD34 + and ALDHbr HSPC content up to 3-fold compared to control. In addition, human CD133 + HSPC cell content was increased to 5-fold following treatment with Pluripotin. Intriguingly, we find that Pluripotin treatment inversely changes bone marrow-derived mesenchymal stem cell (MSC) proliferation and lowers fibroblast growth although there is no effect on adipose-derived MSCs. CHIR-99021 treatment did not have any effect in proliferation of MSCs or fibroblasts. In conclusion, Pluripotin-induced stem cell expansion is unique to HSPCs that can be used to expand HSPCs and reduce unwanted fibroblast or MSC growth in the primary ex vivo cultures.

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Section: Lineage Negative Cell Isolationmentioning

confidence: 99%

“…The plated cells were treated with small molecules of CHIR-99021 and Pluripotin at concentrations of 0.1 μM, 1 μM and 10 μM and DMSO as a control. Dimethyl sulfoxide (0.5 percent) was treated with the cells used as control (Calbiochem, 317275) (Uslu et al, 2020).…”

Section: Umbilical Cord Blood Mononuclear Cell Isolation and Treatmentmentioning

confidence: 99%

Pluripotin facilitates the expansion of hematopoietic stem cells, but restricts the growth of fibroblasts and the proliferation of mesenchymal stem cells from the bone marrow

Turan

Kocabaş

2021

Preprint

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“…UCB and mPB mononuclear cells were isolated by using Ficoll-Paque (HistopaqueTM, Sigma, Cat.No:10831) density gradient centrifugation (1,083g/ml) method in 24 hours post obtaining as we have done previousy (12).…”

Section: Isolation and Culture Of Mncs And Cd34 + Cellsmentioning

confidence: 99%

“…After that, the cells were plated in methylcellulosecontaining medium (MethoCult™ GF M3434, Stemcell Technologies, Cat.No:03444) at a density of 20,000 cells per well in 6-well plate, performed in triplicate. After 14 days, colonies were classi ed as CFU-GEMM, CFU-G/M, BFU-E and B cell colonies and counted by light microscopy as we have done previously (12).…”

Section: Colony Forming Unit (Cfu) Assaysmentioning

confidence: 99%

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Screening of Hematopoietic Small Molecules Facilitates the Establishment of a Chemically Defined Hsc Expansion Medium With Enhanced Performance

Albayrak¹,

Akgöl²,

Turan³

et al. 2021

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Hematopoietic stem cells (HSC), known for their ability to multipotent and self-renew, are often used in HSC transplantation for the treatment of hematological diseases and malignancies. Umbilical cord blood (UCB) and mobilized peripheral blood (mPB), which are alternatives to bone marrow (BM) for HSC transplantation, have reduced HSC. To address this restriction, the ex vivo expansion of HSCs is a highly promising therapeutic approach rather than the use of a double-cord blood unit that induces delays in hematopoietic recovery. We have previously shown that knockout of HSC quiescence genes could increase the HSC pool in vivo. Thus, we thought that targeting HSC quiescence regulators using small molecules could be used for ex vivo expansion of both mPB and UCB-HSC. The goal was to identify novel hematopoietic small molecules (HSMs) and their combinations, and to enhance performance of human HSC expansion medium. We identified and analyzed 35 possible HSMs targeting HSC quiescence factors. We assessed their impact on human HSPC activity, including expansion, quiescence, multilineage capacity, cycling capability and metabolism. We have also investigated their cytotoxic and genotoxic effects on human HSPCs. On the other hand, a transplantation study was performed on immunocompromised mice for the evaluation of the repopulation and engraftment capacities of the expanded cells. We observed that BML-260 and TAME molecules robustly increased both the mPB and UCB-HSPC content and activated the re-entry of HSCs into the cell cycle. Colony Forming Unit (CFU) assay confirmed their improved multilineage capacity. BML-260 proved safer for the viability of expanded cells based on cytotoxicity and genotoxicity assays. In addition, gene expression analysis showed that BML-260 and TAME molecules contributed to HSC expansion by modulating cell cycle kinetics, including p27, Skp2 and Cdh1. In conjunction with these in vitro results, we have observed that BML-260-expanded HSCs had a strong hematopoietic reconstitution capacity. After the determination of the most effective molecule as BML-260, a comparative study of chemically defined media, including various supplements, was analyzed in addition to the BML-260 molecule. These results from in vitro and xenotransplantation experiments have shown that BML-260 molecules can be used therapeutically for human HSC expansion and regulation of HSC activity. In addition, the medium composition found may be a novel platform for human HSC expansion to used in clinical trials.

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BML‐260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability

Albayrak

Akgöl

Turan

et al. 2022

J of Cellular Biochemistry

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Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity.A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-L-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

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Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Cited by 25 publications

References 92 publications

Pluripotin facilitates the expansion of hematopoietic stem cells, but restricts the growth of fibroblasts and the proliferation of mesenchymal stem cells from the bone marrow

Pluripotin facilitates the expansion of hematopoietic stem cells, but restricts the growth of fibroblasts and the proliferation of mesenchymal stem cells from the bone marrow

Screening of Hematopoietic Small Molecules Facilitates the Establishment of a Chemically Defined Hsc Expansion Medium With Enhanced Performance

BML‐260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability

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