Presence and characteristics of circulating megakaryocyte progenitor cells in human fetal blood

Abstract The in vitro growth of early (burst-forming unit-megakaryocyte [BFU- meg]) and late (colony-forming unit-megakaryocyte [CFU-meg]) megakaryocyte progenitors was investigated in midtrimester human fetal blood and compared with adult bone marrow. Most of the experiments were performed in a serum-free fibrin-clot assay, using purified hematopoietic progenitor (CD34+) cells. High BFU-meg and CFU-meg levels were found in human fetal blood, with a clear prevalence of BFU-meg (BFU-meg:CFU-meg ratio, 2.5:1), at variance with adult bone marrow, in which mature CFU-meg predominate (BFU-meg:CFU-meg ratio, 0.6:1). Fetal and adult megakaryocyte progenitors had a similar phenotypic profile for the expression of CD34, HLA-DR, and glycoprotein-complex IIB-IIIA. However, fetal BFU-meg were larger in size (number of megakaryocytic elements per colony) than adult BFU-meg, but were usually composed by only one or two foci of development. On the other hand, fetal and adult CFU-meg were similar in both morphology and size. Fetal megakaryocyte progenitors appeared earlier in culture and had an increased proliferative activity as demonstrated by the higher number of megakaryocyte progenitors in S phase with respect to adult CFU-meg and BFU-meg. Finally, fetal megakaryocyte progenitors displayed a higher sensitivity to stimulatory cytokines, in particular recombinant interleukin-3, than adult megakaryocyte progenitors, whereas they were inhibited by purified transforming growth factor-beta 1 in a similar fashion to adult megakaryocyte progenitors.

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Presence and characteristics of circulating megakaryocyte progenitor cells in human fetal blood

Blood ◽

10.1182/blood.v81.2.385.bloodjournal812385 ◽

1993 ◽

Vol 81 (2) ◽

pp. 385-390 ◽

Cited By ~ 1

Author(s):

G Zauli ◽

L Valvassori ◽

S Capitani

Keyword(s):

Bone Marrow ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Fibrin Clot ◽

Fetal Blood ◽

Phenotypic Profile ◽

Adult Bone Marrow ◽

Adult Bone ◽

Megakaryocyte Progenitors

The in vitro growth of early (burst-forming unit-megakaryocyte [BFU- meg]) and late (colony-forming unit-megakaryocyte [CFU-meg]) megakaryocyte progenitors was investigated in midtrimester human fetal blood and compared with adult bone marrow. Most of the experiments were performed in a serum-free fibrin-clot assay, using purified hematopoietic progenitor (CD34+) cells. High BFU-meg and CFU-meg levels were found in human fetal blood, with a clear prevalence of BFU-meg (BFU-meg:CFU-meg ratio, 2.5:1), at variance with adult bone marrow, in which mature CFU-meg predominate (BFU-meg:CFU-meg ratio, 0.6:1). Fetal and adult megakaryocyte progenitors had a similar phenotypic profile for the expression of CD34, HLA-DR, and glycoprotein-complex IIB-IIIA. However, fetal BFU-meg were larger in size (number of megakaryocytic elements per colony) than adult BFU-meg, but were usually composed by only one or two foci of development. On the other hand, fetal and adult CFU-meg were similar in both morphology and size. Fetal megakaryocyte progenitors appeared earlier in culture and had an increased proliferative activity as demonstrated by the higher number of megakaryocyte progenitors in S phase with respect to adult CFU-meg and BFU-meg. Finally, fetal megakaryocyte progenitors displayed a higher sensitivity to stimulatory cytokines, in particular recombinant interleukin-3, than adult megakaryocyte progenitors, whereas they were inhibited by purified transforming growth factor-beta 1 in a similar fashion to adult megakaryocyte progenitors.

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Human fetal liver MSCs are more effective than adult bone marrow MSCs for their immunosuppressive, immunomodulatory, and Foxp3+ T reg induction capacity

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02176-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yi Yu ◽

Alejandra Vargas Valderrama ◽

Zhongchao Han ◽

Georges Uzan ◽

Sina Naserian ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Immune Responses ◽

Molecular Mechanisms ◽

Fetal Liver ◽

Cytotoxic T Cells ◽

Cell Contact ◽

Adult Bone Marrow ◽

Adult Bone

Abstract Background Mesenchymal stem cells (MSCs) exhibit active abilities to suppress or modulate deleterious immune responses by various molecular mechanisms. These cells are the subject of major translational efforts as cellular therapies for immune-related diseases and transplantations. Plenty of preclinical studies and clinical trials employing MSCs have shown promising safety and efficacy outcomes and also shed light on the modifications in the frequency and function of regulatory T cells (T regs). Nevertheless, the mechanisms underlying these observations are not well known. Direct cell contact, soluble factor production, and turning antigen-presenting cells into tolerogenic phenotypes, have been proposed to be among possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion and activity. We and others demonstrated that adult bone marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4+ helper and CD8+ cytotoxic T cells but also indirectly through the induction of T regs. In parallel, we demonstrated that fetal liver (FL)-MSCs demonstrates much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4+ and CD8+ T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs. Methods MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation, and their proliferation potential. Using different in vitro combinations, we performed co-cultures of FL- or BM-MSCs and murine CD3+CD25−T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs. Results We demonstrated that although both types of MSC display similar cell surface phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4+ and CD8+ murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4+CD25+Foxp3+ T regs compared to BM-MSCs. Conclusions These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction.

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Cytokine production by primary bone marrow megakaryocytes

Blood ◽

10.1182/blood.v84.12.4151.bloodjournal84124151 ◽

1994 ◽

Vol 84 (12) ◽

pp. 4151-4156 ◽

Cited By ~ 56

Author(s):

S Jiang ◽

JD Levine ◽

Y Fu ◽

B Deng ◽

R London ◽

...

Keyword(s):

Bone Marrow ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Interleukin 1 ◽

Human Bone ◽

Human Bone Marrow ◽

Tnf Alpha ◽

Necrosis Factor Alpha ◽

Gm Csf

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.

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Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation

Blood ◽

10.1182/blood.v75.3.596.596 ◽

1990 ◽

Vol 75 (3) ◽

pp. 596-602 ◽

Cited By ~ 112

Author(s):

JR Keller ◽

IK Mcniece ◽

KT Sill ◽

LR Ellingsworth ◽

PJ Quesenberry ◽

...

Keyword(s):

Bone Marrow ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Bone Marrow Cells ◽

Dependent Manner ◽

Tgf Beta ◽

Hematopoietic Progenitor ◽

Growth Factor Beta ◽

Dose Dependent

Abstract We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1- negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5- fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.

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SD-208, a Novel Transforming Growth Factor Beta Receptor I Kinase Inhibitor, Can Stimulate Hematopoiesis in Myelodysplastic Syndrome Progenitors.

Blood ◽

10.1182/blood.v106.11.3448.3448 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3448-3448

Author(s):

Amit Verma ◽

Tony A. Navas ◽

Jing Ying ◽

Aaron N. Nguyen ◽

Perry Pahanish ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Mononuclear Cells ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Specific Inhibitor ◽

Low Grade ◽

Bone Marrow Biopsies

Abstract Transforming Growth Factor β (TGF-β) is a myelosuppressive cytokine that has been implicated in the ineffective hematopoiesis seen in myelodysplastic syndromes (MDS). Overactivation of TGF-β signaling in this disease was demonstrated immunohistochemically by significantly higher nuclear SMAD2 phosphorylation observed in 20 MDS bone marrows when compared with 7 non MDS anemic controls (P < 0.0001, 2 Tailed T Test, Image Pro Plus software). This data along with high levels of membrane-bound and plasma TGF-β observed in MDS patients in previous studies support the development of therapeutics targeting the TGF-β signaling pathways in this disease. SD-208 is a novel, potent and specific inhibitor of TGF-β Receptor I (TGFβ-RI) kinase. We demonstrate that SD-208 blocks the phosphorylation of SMAD2 in hematopoietic progenitors which are at the colony forming unit-erythroid (CFU-E) stage of differentiation. SD-208 also abrogates the G0/G1 cell cycle arrest induced by TGF-β in bone marrow progenitors. SD-208 treatment leads to reversal of the myelosuppressive effects of TGF-β on erythroid and myeloid colony formation from primary human CD34+ cells. Selectivity of SD-208 in inhibiting TGF-β-mediated effects on hematopoiesis was supported by similar results observed with siRNAs targeting SMAD2, a major component of the TGF-b signaling pathway. Finally, the efficacy of SD-208 in MDS was evaluated by treating bone marrow mononuclear cells from 15 patients with early low grade MDS. SD-208 treatment led to dose-dependent increases in erythroid and myeloid colonies after 14 days of in vitro culture. The effect was most notable in patients with high levels of activated SMAD-2, as assessed by immunohistochemical staining of bone marrow biopsies. Stimulation of hematopoiesis in MDS-derived marrow culture by SD-208 demonstrates a novel concept and potential therapeutic role for TGFβ-RI inhibition in this disease. Supported by VISN-17 grant, Harris Methodist Foundation Grant and ASCO YIA to AV

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Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro

Neuroreport ◽

10.1097/00001756-200207020-00023 ◽

2002 ◽

Vol 13 (9) ◽

pp. 1185-1188 ◽

Cited By ~ 113

Author(s):

Byoung J. Kim ◽

Jeong H. Seo ◽

James K. Bubien ◽

Young S. Oh

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Stem Cells ◽

Adult Bone Marrow ◽

Adult Bone

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Use of mesenchymal stem cells from adult bone marrow for injured tissue repair

Orvosi Hetilap ◽

10.1556/oh.2009.28666 ◽

2009 ◽

Vol 150 (27) ◽

pp. 1259-1265 ◽

Cited By ~ 5

Author(s):

Antal Salamon ◽

Erzsébet Toldy

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Transforming Growth Factor Beta ◽

Tissue Repair ◽

Transforming Growth Factor ◽

Injured Tissue ◽

Growth Factor Beta ◽

Adult Bone

A csontvelőből származó mesenchymalis őssejtek pluripotensek, s képesek porc, csont, valamint adiposus és ínsejtekké differenciálódni. Ezen mesenchymalis progenitor sejteket stromasejteknek vagy mesenchymalis őssejteknek nevezik. A csontvelőben két fő sejttípus van: haematopoeticus sejt és stromasejt. Mesenchymalis őssejtek kis beavatkozással nyerhetők a csontvelőből, majd sejtkultúrában szaporíthatóak. Differenciálódásuk bioaktív molekulákkal, specifikus növekedési faktorokkal segíthető elő. A transforming growth factor beta (TGF-β) család tagjai proteinek, közülük a bone morphogenetic proteinek (BMP) a legfontosabb faktorok, amelyek elősegítik a mesenchymalis őssejtek porc- és csontszövetté történő differenciálódását. Kevésbé ismert még ezen sejteknek a tenogenesisben való szerepe, de már vannak biztató adatok e téren is. A mesenchymalis őssejteknek és növekedési faktoroknak a sérült szövetekbe való juttatásra vivő vázanyagra (carrier, scaffold) van szükség. Mesenchymalis őssejtek használhatók fel génterápiára és a tissue engineering alkalmazására. A szerzők jelen munkájukban áttekintik a mesenchymalis őssejtek, biomolekulák és növekedési faktorok szövetpótlás céljából történő használatával foglalkozó kísérletes vizsgálatok eddigi eredményeit és ismertetik a klinikai alkalmazás lehetőségeit.

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Characterization of MSC Potential To Treat GVHD Using Molecular Markers Linked to MSC-Mediated Immunosuppression In Vitro.

Blood ◽

10.1182/blood.v106.11.4322.4322 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4322-4322 ◽

Cited By ~ 3

Author(s):

Diane Carter ◽

Alicia Tyrell ◽

Simon Bubnic ◽

Michelle Marcelino ◽

Keren Kedzierski ◽

...

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Enzyme Activity ◽

Lymphocyte Proliferation ◽

Tryptophan Depletion ◽

Solid Organ ◽

Adult Bone Marrow ◽

Tnf Α ◽

Adult Bone

Abstract Mesenchymal stem cells (MSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage and fat. The biological activities of MSCs suggest a number of potential clinical applications, where each particular application is related to a specific MSC activity mediated by a different mechanism. Osiris Therapeutics has developed a technology for isolation and expansion of hMSCs from adult bone marrow for clinical use. Data from pre-clinical and clinical studies suggest that the ability of MSCs to migrate to inflammatory sites, modulate immune response, down-regulate inflammation, and accelerate tissue repair in the local environment may have therapeutic effects. Therefore in developing therapeutic applications, the MSCs should be verified to display one or more of above-mentioned functions, calling for the need to develop predictive functional assays. Modulation of the immune response is an apparent in vivo therapeutic property of the MSC necessary for successful Graft versus Host Disease (GVHD) treatment. Based on previous knowledge regarding mechanisms underlying MSC-mediated immunosuppressive effects, several markers for developing an MSC potency assay have been proposed. In the present study a relationship between selected markers and hMSC-mediated immunosuppression was investigated in vitro. Results show that co-culture of hMSCs with anti-CD3/CD28-activated peripheral blood mononuclear cells (hPBMCs) caused inhibition of lymphocyte proliferation. The hMSC effect on lymphocyte proliferation is dose-dependent, causing > 50% inhibition at approximately 1:10–1:25 MSC: T-lymphocyte ratio. Supernatants of parallel co-cultures taken on days 1, 3, and 5 were analyzed for prostaglandin 2 (PGE2), tumor necrosis factor-α (TNF-α), and tryptophan. The results showed increased levels of PGE2, decreased levels of TNF-α and increased depletion of tryptophan related to indoleamine 2,3-dioxygenase (IDO) enzyme activity, associated with increasing number of MSCs in each well. The quantity of PGE2 on day 1 and the level of tryptophan on day 5 in the MSC-PBMC co-culture supernatants correlated to the level of inhibition of proliferation, with the PGE2 range from approximately 11,000 to 22,000 pg/mL and 50% tryptophan depletion resulting in a 50% inhibition of the lymphocyte proliferation point. Further studies demonstrated that the addition of TNF-α to MSCs induced PGE2 secretion at a level which was similar to that detected in the co-culture studies of MSCs-PBMC. Thus, a strong correlation between inducible PGE2 secretion/IDO enzyme activity and the inhibition of lymphocyte proliferation by hMSCs in vitro indicates key molecules responsible for hMSC functional activity related to the immunological responses involved with diseases such as GVHD, solid organ transplantation and autoimmune diseases.

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