In Vitro Proliferation and Differentiation of Megakaryocytic Progenitors in Patients with Aplastic Anemia, Paroxysmal Nocturnal Hemoglobinuria, and the Myelodysplastic Syndromes

Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34+ cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulinlike growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 103fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34+ cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34+-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.

Download Full-text

In Vitro Proliferation and Differentiation of Human Hair Follicle Stem Cells on Chitosan-Skin Regenerating Template

Current Tissue Engineering ◽

10.2174/2211542005666151123203226 ◽

2015 ◽

Vol 05 (999) ◽

pp. 1-1

Author(s):

Abu Bakar Mohd Hilmi ◽

Mohd Noor Norhayati ◽

Ahmad Sukari Halim ◽

Chin Keong Lim ◽

Zulkifli Mustafa ◽

...

Keyword(s):

Stem Cells ◽

Hair Follicle ◽

Human Hair ◽

Human Hair Follicle ◽

In Vitro Proliferation ◽

Proliferation And Differentiation ◽

Hair Follicle Stem Cells ◽

Follicle Stem Cells

Download Full-text

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Brazilian Journal of Medical and Biological Research ◽

10.1590/1414-431x20132620 ◽

2013 ◽

Vol 46 (8) ◽

pp. 681-688 ◽

Cited By ~ 4

Author(s):

Z. Li ◽

J. Chen ◽

L. Li ◽

J.H. Ran ◽

J. Liu ◽

...

Keyword(s):

Oval Cells ◽

In Vitro Proliferation ◽

Potential Capacity ◽

Hepatic Oval Cells ◽

Proliferation And Differentiation

Download Full-text

Apoptosis Resistance of Blood Cells From Patients With Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v90.7.2716.2716_2716_2722 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2716-2722 ◽

Cited By ~ 2

Author(s):

Kentaro Horikawa ◽

Hideki Nakakuma ◽

Tatsuya Kawaguchi ◽

Norihiro Iwamoto ◽

Shoichi Nagakura ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Clinical Manifestations ◽

Interferon Γ ◽

Apoptosis Resistance ◽

Molecular Events ◽

Cd34 Cells ◽

Factor Α

Bone marrow (BM) hypoplasia is a major cause of death in paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about the molecular events leading to the hypoplasia. Considering the close pathologic association between PNH and aplastic anemia (AA), it is suggested that a similar mechanism operates in the development of their BM failure. Recent reports have indicated apoptosis-mediated BM suppression in AA. It is thus conceivable that apoptosis also operates to cause BM hypoplasia in PNH. If this is the case, PNH clones need to survive apoptosis and show considerable expansion leading to clinical manifestations. We report here that granulocytes obtained from 11 patients with PNH were apparently less susceptible than those from 20 healthy individuals to both spontaneous apoptosis without any ligands and that induced by anti-FAS (CD95) antibody in vitro. The patients' BM CD34+ cells were also resistant to apoptosis induced by treatment with tumor necrosis factor-α, interferon-γ, and subsequently with anti-FAS antibody. In lymphocytes, the pathologic resistance was not discriminated from inherent resistance to apoptosis. Granulocytes from 13 patients with AA and 12 patients with myelodysplastic syndrome (MDS) exhibited similar resistance to apoptosis. CD34+ cells from MDS-BM also showed similar tendency. Thus, the comparative resistance to apoptosis supports the pathogenic implication of apoptosis in marrow injury of PNH and related stem cell disorders.

Download Full-text

Impaired Hematopoiesis in Paroxysmal Nocturnal Hemoglobinuria/Aplastic Anemia Is Not Associated With a Selective Proliferative Defect in the Glycosylphosphatidylinositol-Anchored Protein-Deficient Clone

Blood ◽

10.1182/blood.v89.4.1173 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1173-1181 ◽

Cited By ~ 84

Author(s):

Jaroslaw P. Maciejewski ◽

Elaine M. Sloand ◽

Tadatsugu Sato ◽

Stacie Anderson ◽

Neal S. Young

Keyword(s):

Protein Expression ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Surface Proteins ◽

In Vitro Assays ◽

Normal Numbers ◽

Normal Volunteers ◽

Cd34 Cells ◽

Cd59 Expression

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI− phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+CD59+ and CD34+CD59− cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+CD59− cells were both CD59− and CD55−. A very small population of CD34+CD59− cells was also detected in some normal volunteers; after sorting, these CD34+CD59− cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A–deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein–deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A– clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.

Download Full-text

Existence of a pool of T-lymphocyte colony-forming cells (T-CFC) in human bone marrow and their place in the differentiation of the T- lymphocyte lineage

Blood ◽

10.1182/blood.v58.5.911.911 ◽

1981 ◽

Vol 58 (5) ◽

pp. 911-915 ◽

Cited By ~ 26

Author(s):

F Triebel ◽

WA Robinson ◽

AR Hayward ◽

PG Goube de Laforest

Keyword(s):

Bone Marrow ◽

T Cell ◽

T Lymphocyte ◽

Cell Lineage ◽

Marrow Graft ◽

In Vitro Proliferation ◽

Complement Dependent Cytotoxicity ◽

Self Tolerance ◽

Proliferation And Differentiation

Abstract The existence and characteristics of bone marrow T-cell progenitors have not yet been established in man. Several pieces of evidence such as the reconstitution of certain immunodeficiencies by bone marrow graft suggest that T-cell precursors are present in the bone marrow. We report the growth of T-cell colonies from bone marrow populations using PHA-stimulated lymphocyte-conditioned medium containing T-cell growth factor (TCGF). Rosetting experiments and complement-dependent cytotoxicity assays with monoclonal antibodies indicate that the bone marrow T colony-forming cells (T-CFC) are E- OKT 3- and la+, i.e., immature progenitors. The colonies derived from these cells have the phenotype of mature T cells: E + OKT 3 + la- with either helper (OKT 4+) and suppressor (OKT 8 +) antigens. These results suggest that a thymic microenvironment may not be necessary for the in vitro proliferation and differentiation of the T-cell lineage in adult humans. These methodologies may permit direct investigation of early phenomena concerning the T-cell lineage, such as the acquisition of self-tolerance, the formation of a repertoire of specificities, and the HLA restriction phenomena that we believe takes place before the thymic maturation.

Download Full-text

In vitro proliferation and differentiation of canine bone marrow derived mesenchymal stem cells over hydroxyl functionalized CNT substrates

Biotechnology Reports ◽

10.1016/j.btre.2019.e00387 ◽

2019 ◽

Vol 24 ◽

pp. e00387

Author(s):

A.P. Madhusoodan ◽

Kinsuk Das ◽

Bhabesh Mili ◽

Kuldeep Kumar ◽

Ajay Kumar ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

In Vitro Proliferation ◽

Proliferation And Differentiation

Download Full-text

Common and pre-B acute lymphoblastic leukemia cells express interleukin 2 receptors, and interleukin 2 stimulates in vitro colony formation

Blood ◽

10.1182/blood.v66.3.556.556 ◽

1985 ◽

Vol 66 (3) ◽

pp. 556-561 ◽

Cited By ~ 44

Author(s):

I Touw ◽

R Delwel ◽

R Bolhuis ◽

G van Zanen ◽

B Lowenberg

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Colony Formation ◽

B Lymphocyte ◽

Lymphoblastic Leukemia ◽

Interleukin 2 ◽

Colony Growth ◽

Leukemic Cells ◽

In Vitro Proliferation ◽

Proliferation And Differentiation

Abstract The role of interleukin 2 (IL 2) as a possible regulator of in vitro proliferation and differentiation of non-T acute lymphoblastic leukemia (ALL) cells was investigated. For this purpose, leukemic cells from the blood or bone marrow of eight untreated patients with common or pre-B ALL were analyzed using the anti-Tac monoclonal antibody (reactive with the IL 2 receptor) in indirect immunofluorescence. The receptors for IL 2, which were initially absent from the cell surface, were induced on high percentages of the ALL cells after the in vitro exposure to the lectin phytohemagglutinin or the phorbol ester 12-O- tetradecanoylphorbol-13-acetate in six patients, suggesting that the cells had become sensitive to IL 2. In colony cultures to which feeder leukocytes and IL 2 had been added, colony growth was obtained in five of eight cases. Whereas the cells from one patient formed colonies in the absence of exogenous stimuli, the cells from others were dependent on the addition of feeder leukocytes plus IL 2. In the latter cases, feeder leukocytes alone, releasing some IL 2, stimulated growth suboptimally at different cell concentrations. Their stimulative effect was significantly enhanced when leukocyte-derived IL 2 or pure recombinant IL 2 was supplemented. Alone, IL 2 (up to 500 U/mL) did not support colony formation. Apparently, IL 2 and feeder leukocytes are both required for the induction of colonies in these cases of ALL. From cell sorting of fluorescent anti-common ALL antigen (CALLA) stained cells it appeared that colonies descended from cells with high as well as low or negative CALLA expression. Immunophenotyping demonstrated the presence of the original leukemia markers on colony cells, but was not indicative of maturation of ALL toward more differentiated B cells. We suggest that IL 2 can stimulate the in vitro proliferation of certain neoplastic B lymphocyte progenitors.

Download Full-text