307 REPROGRAMMING OF PIG SOMATIC CELLS TO PLURIPOTENCY WITH SLEEPING BEAUTY TRANSPOSON VECTORS CONTAINING THE PORCINE TRANSCRIPTION FACTOR SEQUENCES

2013 ◽  
Vol 25 (1) ◽  
pp. 300
Author(s):  
S. Petkov ◽  
M. Nowak-Imialek ◽  
P. Hyttel ◽  
H. Niemann
Keyword(s):  
Transcription Factor ◽  
Retroviral Vectors ◽  
Culture Conditions ◽  
Sleeping Beauty ◽  
Patient Specific ◽  
Differentiation Potential ◽  
Serum Replacement ◽  
Gfp Reporter ◽  
Fetal Fibroblasts

Induced pluripotent stem cells (iPSC), developed by Yamanaka and co-workers (Takahashi et al., 2006), hold significant potential for the development of regenerative therapies due to the possibilities of deriving patient-specific pluripotent cells. In this aspect, the pig is an important animal model for testing iPSC-based applications for the human medicine. However, even though significant progress has been made, the derivation of porcine iPSC lines fully equivalent to those from mouse and human has been elusive. To date, most of the reported putative pig iPSC lines have been derived with the use of lentiviral or retroviral vectors harboring the mouse or human transcription factor sequences. Here, we report the construction of Sleeping Beauty (SB) transposon vectors with porcine cDNA sequences coding for OCT4, SOX2, NANOG, C-MYC, and KLF4, in addition to the human LIN28. By using standard cloning techniques, we produced 2 polycistronic SB-CAG-pOSMK-ires-Tomato and SB-Ef1a-pNANOG-ires-hLIN28 transposon vectors and we transfected them together with the SB100X transposase into pig fetal fibroblasts (pFF) harboring a mouse OCT4-GFP reporter construct (Nowak-Imialek et al., 2010). Both the basic transposon and transposase vectors were generously provided by Dr. Zoltan Ivics from Paul Ehrlich Institute, Langen, Germany. In each experiment, 2 × 106 pFF were electroporated with 3 µg of each transposon together with 0.5 µg of SB100X. Two days after transfection, the cells were transferred to mouse embryonic fibroblast (MEF) feeders and cultured with iPSC medium [DMEM with antibiotics, nonessential amino acids, 20% Knockout serum replacement, 5 ng mL–1 human recombinant basic fibroblast growth factor (bFGF), and 1000 U mL–1 ESGRO]. Two weeks post-transfection, multiple compact colonies were apparent (mean = 2195; SEM = 166; n = 3), which were 95% alkaline phosphatase-positive and ~80% expressed the OCT4-GFP reporter. Reverse transcription-PCR showed that these colonies expressed high levels of endogenous OCT4, SOX2, NANOG, REX1, UTF1, CDH1, and TDH. The cultures were passaged by trypsin disaggregation, followed by seeding on fresh feeders at density 10 × 103 cells cm–2. The established cell lines proliferated as compact, mouse iPSC-like colonies that retained their OCT4 reporter expression as well as the expression of the endogenous pluripotency genes for at least 30 passages. The expression of the transgenes was persistent and showed that no silencing had occurred, even in long-term culture. When subjected to in vitro differentiation protocols, the putative iPSC formed mainly large trophectodermal (TE) vesicles (positive for TE markers CDX2, PAG, and HAND1), fibroblast-like, and neuronal-like cells. These cells still expressed the transgenes as well as most endogenous pluripotency markers, demonstrating limited differentiation capacity. Because the stable transgene expression and the suboptimal culture conditions are the most likely causes of this limited differentiation potential, we are currently working on generating transgene-free iPSC lines under improved cell culture conditions.

scholarly journals Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides

2021 ◽  
Vol 22 (6) ◽  
pp. 2820
Author(s):  
Stephan Altmann ◽  
Jürgen Mut ◽  
Natalia Wolf ◽  
Jutta Meißner-Weigl ◽  
Maximilian Rudert ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Adipogenic Differentiation ◽  
Culture Conditions ◽  
Surface Proteins ◽  
Differentiation Potential ◽  
Or Gene ◽  
Mannose Derivatives ◽  
Metabolic Glycoengineering ◽  
Scientific Questions

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

scholarly journals 34 LIFESPAN AND CHROMOSOMAL STABILITY OF BOVINE AND PORCINE FETAL FIBROBLAST CELLS CULTURED IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
A.M. Giraldo ◽  
J.W. Lynn ◽  
C.E. Pope ◽  
R.A. Godke ◽  
K.R. Bondioli
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Cycle Duration ◽  
Fibroblast Cells ◽  
Proliferative Capacity ◽  
Chromosomal Stability ◽  
Fetal Fibroblasts ◽  
Fetal Bovine

The low efficiency of nuclear transfer (NT) has been related to factors such as mitochondria heteroplasmy, failure of genomic activation, and asynchrony between the donor karyoplast and recipient cytoplast. Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. It is known that suboptimal culture conditions can induce chromosomal abnormalities, and the use of aneuploid donor cells during NT can lead to a high incidence of abnormal cloned embryos (Giraldo et al. 2004 Reprod. Fertil. Dev. 16, 124 abst). The purpose of this study was to determine the lifespan and chromosomal stability of bovine and porcine fetal cells. Four bovine and four porcine fibroblast cells lines were established from 50-day and 40-day fetuses, respectively. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in 5% CO2. Each cell line was passaged to senescence. Total population doublings (PDs) and cell cycle duration were calculated. To determine the chromosome numbers at different PDs, cells were synchronized in metaphase, fixed, and stained. ANOVA and chi-square tests were used to analyze differences in PDs and proportion of aneuploid cells between cell lines, respectively (P < 0.05). The results show that proliferative capacity was not different between cell lines derived from the same species. Cell lines derived from bovine and porcine fetuses had different in vitro lifespans (33 PDs vs. 42 PDs, respectively; P < 0.05). The mean length of the cell cycles for both bovine and porcine fetal fibroblasts was ∼28 h. The percentage of aneupliod cells in both bovine and porcine fetal cell lines increased progressively with duration of culture (see Table) and was high throughout the study. The proliferative capacity of cultured cells was similar within individuals of the same species, but growth characteristics differed between fetal bovine and porcine cell lines. The progressive increase of aneuploid cells could be due to suboptimal culture conditions or unusual chromosome instability in the particular fetuses used. These data demonstrate the importance of determining chromosome content and the use of cells at early passages to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of NT.

scholarly journals Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-15
Author(s):  
Audrey Chabrat ◽  
Emmanuelle Lacassagne ◽  
Rodolphe Billiras ◽  
Sophie Landron ◽  
Amélie Pontisso-Mahout ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Neurodegenerative Diseases ◽  
Dopaminergic Neurons ◽  
Adult Stem Cells ◽  
Morphological Changes ◽  
Direct Conversion ◽  
Patient Specific ◽  
Induced Pluripotent

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson’s disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

Methylation of RUNX1 by the Nuclear PRMT5/COPR5 Complex Regulates Its Cellular Location in Leukemia Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3403-3403
Author(s):  
Xinyang Zhao ◽  
Ly P. Vu ◽  
Fabiana Perna ◽  
Fan Liu ◽  
Hao Xu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Arginine Methylation ◽  
Binding Domain ◽  
Dependent Manner ◽  
Dna Binding Domain ◽  
Sm Proteins ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Abstract 3403 RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture

2014 ◽  
Vol 306 (4) ◽  
pp. C322-C333 ◽  
Author(s):  
Andrea Zaniboni ◽  
Chiara Bernardini ◽  
Marco Alessandri ◽  
Chiara Mangano ◽  
Augusta Zannoni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Vitro Culture ◽  
Umbilical Vein ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Tunica Media ◽  
Large Numbers ◽  
Umbilical Vein Endothelial Cells ◽  
Pig Animal Model

Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

scholarly journals Human Tendon Stem/Progenitor Cell Features and Functionality Are Highly Influenced by in vitro Culture Conditions

2021 ◽  
Vol 9 ◽  
Author(s):  
Carlotta Perucca Orfei ◽  
Annie C Bowles ◽  
Dimitrios Kouroupis ◽  
Melissa A Willman ◽  
Enrico Ragni ◽  
...  
Keyword(s):  
Adipogenic Differentiation ◽  
Tendon Repair ◽  
Culture Conditions ◽  
Cellular Level ◽  
Immunomodulatory Activity ◽  
Differentiation Potential ◽  
Culture Density ◽  
Inflammatory Stimuli ◽  
Human Tendon

Our understanding of tendon biology continues to evolve, thus leading to opportunities for developing novel, evidence-based effective therapies for the treatment of tendon disorders. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. We described different molecular and phenotypic profiles of TSPCs modulated by culture density, as well as their multipotency and secretory activities. Moreover, in the same experimental setting, we evaluated for different responses to inflammatory stimuli mediated by TNFα and IFNγ. We also preliminarily investigated their immunomodulatory activity and their role in regulating degradation of substance P. Our findings indicated that TSPCs cultured at low density (LD) exhibited cobblestone morphology and a reduced propensity to differentiate. A distinctive immunophenotypic profile was also observed with high secretory and promising immunomodulatory responses when primed with TNFα and IFNγ. In contrast, TSPCs cultured at high density (HD) showed a more elongated fibroblast-like morphology, a greater adipogenic differentiation potential, and a higher expression of tendon-related genes with respect to LD. Finally, HD TSPCs showed immunomodulatory potential when primed with TNFα and IFNγ, which was slightly lower than that shown by LD. A shift from low to high culture density during TSPC expansion demonstrated intermediate features confirming the cellular adaptability of TSPCs. Taken together, these experiments allowed us to identify relevant differences in TSPCs based on culture conditions. This ability of TSPCs to acquire distinguished morphology, phenotype, gene expression profile, and functional response advances our current understanding of tendons at a cellular level and suggests responsivity to cues in their in situ microenvironment.

53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
Y. U. Kim ◽  
D. P. Bhandari ◽  
M. S. Hossein ◽  
S. M. Park ◽  
E. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Differential Staining ◽  
In Vitro Production ◽  
Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P &lt; 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P &lt; 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

29 SCRIPTAID IMPROVES SOMATIC NUCLEAR TRANSFER EFFICIENCY DURING IN VITRO CULTURE OF PORCINE EMBRYOS DERIVED FROM INBRED MINIATURE PIG FETAL FIBROBLASTS

2015 ◽  
Vol 27 (1) ◽  
pp. 107
Author(s):  
R. Koppang ◽  
N. R. Mtango ◽  
M. Barcelo-Fimbres ◽  
J. P. Verstegen
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
Treated Group ◽  
Culture Conditions ◽  
Control Group ◽  
Miniature Pig ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Porcine somatic cell nuclear transfer (SCNT) is limited to the same or next day surgical embryo transfer due to poor culture conditions in vitro. In this study, we aimed to overcome this problem by treating SCNT embryos with scriptaid, an inhibitor of histone deacetylase (HDACi) that helps with epigenetic reprogramming of the somatic nuclei. Scriptaid was chosen over other HDACi because it has been shown to improve histone acetylation in the same pattern as that of IVF embryos as well as its low toxicity characteristic (Zhao et al. 2009 Biol. Reprod. 81, 525–530; Zhao et al. 2010 Cell Reprogram. 12, 75–78). An inbred miniature pig fetal fibroblast cell line that is known to give low blastocyst rate in culture was used as a source of donor cells transferred into enucleated oocytes. Traditional SCNT was performed; embryos were fused and chemically activated in 10 µM ionomycin for 5 min and 2 mM DMAP for 3 to 4 h before being transferred into scriptaid. Embryos were treated with 500 nM scriptaid (Zhao et al. 2010) for 18 h and the untreated group was used as control. A total of 806 oocytes were used in 8 replicates. The constructed embryos were cultured in modified porcine zygote medium 5 (mPZM-5) for 7 days at 39°C in 5% O2, 5% CO2, 90% N2 atmosphere. Cleavage rates were assessed at 2.5 days and blastocyst rates at Day 7 after activation. Data were analysed by ANOVA using GLM, and percentages were transformed using arcsin square root using Statistix 10 software (Tallahassee, FL, USA). There were no differences in cleavage rates for control group v. scriptaid (55.3 v. 49.9%; P > 0.1; Table 1). The blastocyst rate per construct showed remarkable increase in the scriptaid treated group compared with the control group (12.8 v. 2.2%; P < 0.01; Table 1). Similarly, a significant effect was observed for blastocyst per embryos cleaved where scriptaid had higher rates compared with control (25.8 v. 5.8%; P < 0.01). These results indicated that improving nuclear reprogramming of miniature porcine SCNT clones by scriptaid treatment enhanced blastocyst production during the in vitro culture of porcine embryos. Table 1.Mean (± s.e.m.) measures of embryonic development of SCNT embryos

In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3861-3861
Author(s):  
Jun Ooehara ◽  
Hina Takano ◽  
Shin-ichiro Takayanagi ◽  
Hiromitsu Nakauchi ◽  
Hideo Ema
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Marrow Cells

Abstract Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.

Sign in / Sign up

Export Citation Format

Share Document