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Transforming growth factor ?1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy.

ABSTRACT: BACKGROUND:Generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) in vitro takes about 21?days, making it unaffordable for clinical applications. Acceleration of the in vitro erythropoiesis process by using small molecules could eventually make the large-scale production of these cells commercially viable. Transforming Growth Factor ?1 (TGF-?1) has been shown to have a dose-dependent activity on the HSCs: at high concentration it inhibits, whereas at low concentration it stimulates the HSCs growth. At high concentration, it also inhibits erythropoiesis but accelerates terminal erythroid differentiation of cell lines and erythroid progenitors. Here we examined whether the use of low concentration of TGF-?1 would be beneficial for increasing RBC production by stimulating HSC growth and also supporting erythroid differentiation. Such a strategy could make RBC production in vitro more efficient and cost-effective for clinical applications. METHODS:HSCs isolated from Apheresis samples were differentiated into mature RBCs by the sequential addition of specific combinations of growth factors for 21?days. In the control set, only EPO (3?IU/ml) was added whereas, in the test set, TGF-?1 at a concentration of 10?pg/ml was added along with EPO (3?IU/ml) from day 0. RESULTS:We found that a low concentration of TGF-?1 has no inhibitory effect on the proliferation of the early stages of erythropoiesis. Additionally, it significantly accelerates terminal stages of erythroid differentiation by promoting BNIP3L/NIX-mediated mitophagy. CONCLUSIONS:Incorporation of TGF-?1 at 10?pg/ml concentration in the differentiation medium accelerates the in vitro erythropoiesis process by 3?days. This finding could have potential applications in transfusion medicine.

SUBMITTER: Kuhikar R

PROVIDER: S-EPMC7029523 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Transforming growth factor β1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy.

Kuhikar Rutuja R Khan Nikhat N Philip Joseph J Melinkeri Sameer S Kale Vaijayanti V Limaye Lalita L

Stem cell research & therapy 20200219 1

<h4>Background</h4>Generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) in vitro takes about 21 days, making it unaffordable for clinical applications. Acceleration of the in vitro erythropoiesis process by using small molecules could eventually make the large-scale production of these cells commercially viable. Transforming Growth Factor β1 (TGF-β1) has been shown to have a dose-dependent activity on the HSCs: at high concentration it inhibits, whereas at low concentration ...[more]

PMID: 32075694

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Project description:Unlabelled: We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (?) and fetal (?) but not adult (?) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs.SignificanceAs worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.

Project description:BackgroundPrior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation.MethodsWe administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay.ResultsCSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48-CD150+ HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48-CD150+ HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment.ConclusionThese results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

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Transforming growth factor ?1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy.

Publications

Transforming growth factor β1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy.

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