Maintenance of Osteogenic Differentiation Capacity of MSPC Despite Amplified Proliferation Under Elevated Oxgen Conditions

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1916-1916
Author(s):  
Katharina Schallmoser ◽  
Nicole A Hofmann ◽  
Andreas Reinisch ◽  
Anna Ortner ◽  
Claudia Url ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Osteogenic Differentiation ◽  
Malignant Transformation ◽  
Culture Conditions ◽  
Seeding Density ◽  
Oxygen Conditions

Abstract Abstract 1916 Clinical trials are underway to test the safety and efficacy of mesenchymal stem/progenitor cells (MSPCs) in various diseases. Due to their low frequency in situ, MSPC expansion is the prerequisite for dose finding studies as well as for most applications in adult patients. Notably, cultured MSPCs are a mixture of heterogeneous cells in various stages of cell cycle, proliferation and differentiation activity. A major safety concern for MSPC propagation is the risk of malignant transformation or premature senescence hampering MSPC function. The in vitro and consequently in vivo cellular characteristics may be influenced by factors as tissue source, age of the donor, materials and media, growth factors and oxygen pressure, arguing for standardized culture procedures at least in clinical trials. Defining the optimal conditions for efficient expansion of clinical grade cell therapeutics is still a challenge. We have previously shown that long-term expanded human bone marrow-derived MSPCs acquired senescence-related gene expression changes independent of culture conditions (Haematologica 2010). It has been speculated that elevated oxygen (20% air O2) contributes to genomic instability and malignant transformation in vitro. We therefore analyzed the influence of different oxygen conditions during long-term expansion on MSPC behavior focussing osteogenic differentiation. A gene panel previously defined as senescence markers was tested for differential expression after varying culture conditions. Bone marrow-derived MSPCs were expanded in α-MEM supplemented with 10% human platelet lysate replacing fetal bovine serum under physiologic conditions (5% O2) or air oxygen (20% O2) until spontaneous cessation of proliferation. Osteogenic induction was analyzed by Alizarin red. RNA was isolated from corresponding early and late passages and analyzed by qRT-PCR for p16ink4a, PARG1, CDKN2B, PTN and MCM6. In total, MSPCs could be cultured for 5 passages at 30 cells/cm2 and for 10 passages at 3,000 cells/cm2 for up to 85 days resulting in more cumulative population doublings (PDs) of MSPCs at air O2 compared to 5% O2 and in cultures with low compared to standard seeding density. Long-term cultured MSPCs after 40 PDs (air O2) and 35 PDs (5% O2) retained their osteogenic differentiation capacity. Compared to early passages, RT-PCR in late passages revealed an up-regulation of p16ink4a, PARG1 and CDKN2B without specific influence of culture conditions. PTN and MCM6 were significantly down-regulated, mainly in air O2 cultures with high seeding density correlating with diminished cell proliferation compared to low density cultures. There was no evidence of immortalization or malignant transformation. The capacity for in vivo bone formation of long term cultured MSPCs is currently tested in a novel humanized mouse model for bone and marrow niche formation (Blood 2012). Long term expansion of MSPCs under animal serum-free air oxygen conditions was safe and most efficient at low seeding density. Even in late passages (>30 PDs) MSPCs preserved their potential for osteogenic differentiation in vitro. At air oxygen delayed replicative senescence was observed, mainly at low seeding density. There was no evidence for immortalization or transformation indicating applicability of standardized ambient air culture conditions for pre-clinical cell expansion. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acute Myeloid Leukemia (AML) in a 3D Bone Marrow Niche Showed High Performance for in Vitro and In Vivo Drug Screenings

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 544-544
Author(s):  
Giulia Borella ◽  
Ambra Da Ros ◽  
Elena Porcù ◽  
Claudia Tregnago ◽  
Maddalena Benetton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Board Of Directors ◽  
Advisory Committees ◽  
In Vivo Models ◽  
3D Cultures ◽  
Nsg Mice

Chemotherapy still remains the pillar of treatment of children with AML, a disease in which refinements in diagnostic approaches, minimal residual disease monitoring, and patient stratification have resulted into remarkable progresses during the past decade. However, most of the recently tested, novel anti-leukemia agents failed during pre-clinical and clinical validation phases, and one main limit in AML field is the inappropriateness of current preclinical models used to study drug efficacy, this jeopardizing the advance of phase II and III clinical trials, especially for children. In light of this consideration, we aimed at creating novel robust in vitro and in vivo approaches to discover or to re-assess alternative treatments to improve the portfolio of agents active in childhood AML. For this purpose, we developed new protocols for long-term 3D-AML cultures to perform more predictable high throughput drug screening in vitro, and, once identified the best compounds, to create new pre-clinical in vivo models. We set up the bone marrow (BM) endosteal niche by using a biomimetic 3D structure, made up of engineered hydroxyapatite and collagen I, where we seeded mesenchymal stromal cells derived either from AML patients (AML-MSCs) or from healthy BM donors (h-MSCs), together with osteoblasts, endothelial cells and finally AML blasts. We studied AML cell proliferation and clonogenicity cultured in 3D. We obtained results from twenty 3D long-term cultures of different primary AML, confirming blast proliferation up to 21 days. Clonogenic potential and immunophenotype preservation of the original AML blasts was also documented. At the same time, we compared AML-MSCs with h-MSCs, finding that AML-MSCs exhibited a higher proliferation rate (40% increase proliferation at 72 and 96 hours, p<0.001), and commitment to osteogenic differentiation, this latter occurring after 7 days with respect to 21 days of h-MSCs (p<0.01). To better characterize AML-MSCs features supporting AML cell growth, we cultured AML-MSCs and h-MSCs in an inflammatory environment (IL1β, IL6 and TNFα), observing that AML-MSCs did not exert anti-inflammatory activity by HUVEC tube formation assay (n=6, p<0.001). This latter finding was supported by a peculiar secretome profile defined by mass-spectrometry revealing factors as SERPINE1, CHI3L1, TIMP1, and PTX3 being differentially secreted. Thus, a drug targeting of AML-MSCs would be desirable, and we performed a screening of 480 compounds. This screening identified 17/480 active compounds capable of reducing AML-MSCs proliferation without toxicity over h-MSCs and AML blasts. We identified one main compound able to selectively reduce AML-MSCs proliferation, that, when combined to novel therapeutic agents for AML, such as the Quizartinib, I-BET inhibitor and Dasatinib in the 3D cultures, showed a synergistic effect (CI=0.5, p<0.05) towards FLT3-ITD, MLL-rearranged or c-KIT mutated AML. We then proceeded with two pilot studies in order to define the use of this 3D-AML cultures in in vivo setting. We implanted these organoids in the back of NSG mice and monitored leukemia engraftment in the scaffolds, as well as AML dissemination in peripheral blood. We documented the niche being vascularized and well organized by immunohistochemical staining for mCD31, hCD45 and hOPN, whereas we had a low rate of success in AML dissemination out of the niche (1 AML out of 14 implanted in 15 months). On the contrary, we observed that AML cells proliferated in the 3D up to 9 months after implantation, suggesting that this system is more suitable for an easy and quick in loco drug testing. Thus, we implanted AML, after being transduced with luciferase, in the 3D niche in NSG mice and monitored luciferase activity during intra-peritoneal drug treatments. We evaluated some new and old compounds known to target leukemia cells and documented a significant reduction of luciferase in the 3D when a drug was active, supporting our 3D scaffolds as a novel useful in vivo system to screen selected drugs before the prioritization of the best one to be used in a pre-clinical setting in PDXs. In conclusion, our data support the possibility to work with long-term 3D cultures of AML in vitro to identify new drugs, and we attribute to AML-MSCs a crucial supportive role to be further considered in in vivo settings for novel combo strategies. Finally, these findings could help test new compounds to be validated in future clinical trials. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4149-4158 ◽  
Author(s):  
M Trevisan ◽  
XQ Yan ◽  
NN Iscove
Keyword(s):  
Colony Formation ◽  
Interleukin 1 ◽  
Methyl Cellulose ◽  
Culture Conditions ◽  
Liquid Suspension ◽  
Almost All ◽  
Marrow Cells

Abstract This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

scholarly journals Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4102-4109 ◽  
Author(s):  
CI Civin ◽  
G Almeida-Porada ◽  
MJ Lee ◽  
J Olweus ◽  
LW Terstappen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Population ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Fetal Sheep ◽  
Adult Human

Abstract Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

The Use of Erythropoietin in the Treatment of Post-bone Marrow Transplatation Anemia

1993 ◽  
Vol 16 (5_suppl) ◽  
pp. 8-12 ◽  
Author(s):  
A.M. Vannucchi ◽  
A. Bosi ◽  
A. Grossi ◽  
S. Guidi ◽  
R. Saccardi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Bone Marrow Transplantation ◽  
Blood Transfusion ◽  
In Vitro Models ◽  
Immunosuppressive Drug ◽  
Number Of Patients

The issue of the role of erythropoietin (Epo) in the erythroid reconstitution after bone marrow transplantation (BMT) has been addressed in several recent studies. A defective Epo production in response to anemia has been shown to occur in patients undergoing allogeneic BMT unlike in most of those subjected to an autologous rescue. The factors involved in the inadeguate Epo production in BMT are discussed, with particular attention to the role of the immunosuppressive drug cyclosporin-A, which has been shown to inhibit Epo production in both in vivo and in vitro models. The observation of defective Epo production eventually led to the development of clinical trials of recombinant human Epo (rhEpo) administration in BMT patients; the aims of these studies were to stimulate erythroid engraftment, hence reducing blood transfusion exposure. Although the number of patients studied up to now is relatively small, a benefit from rhEpo administration in terms of accelerated erythroid engraftment seems very likely, and it may also be associated with decreased transfusional needs in most treated patients. However, further studies are needed to better define indications, dosages and schedules of rhEpo in BMT patients.

scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

scholarly journals MRI Tracking of SPIO- and Fth1-Labeled Bone Marrow Mesenchymal Stromal Cell Transplantation for Treatment of Stroke

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Xiaolei Huang ◽  
Yang Xue ◽  
Jinliang Wu ◽  
Qing Zhan ◽  
Jiangmin Zhao
Keyword(s):  
Bone Marrow ◽  
Prussian Blue ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Signal Intensity ◽  
Immunofluorescent Staining ◽  
Blue Staining

We aimed to identify a suitable method for long-term monitoring of the migration and proliferation of mesenchymal stromal cells in stroke models of rats using ferritin transgene expression by magnetic resonance imaging (MRI). Bone marrow mesenchymal stromal cells (BMSCs) were transduced with a lentivirus containing a shuttle plasmid (pCDH-CMV-MCS-EF1-copGFP) carrying the ferritin heavy chain 1 (Fth1) gene. Ferritin expression in stromal cells was evaluated with western blotting and immunofluorescent staining. The iron uptake of Fth1-BMSCs was measured with Prussian blue staining. Following surgical introduction of middle cerebral artery occlusion, Fth1-BMSCs and superparamagnetic iron oxide- (SPIO-) labeled BMSCs were injected through the internal jugular vein. The imaging and signal intensities were monitored by diffusion-weighted imaging (DWI), T2-weighted imaging (T2WI), and susceptibility-weighted imaging (SWI) in vitro and in vivo. Pathology was performed for comparison. We observed that the MRI signal intensity of SPIO-BMSCs gradually reduced over time. Fth1-BMSCs showed the same signal intensity between 10 and 60 days. SWI showed hypointense lesions in the SPIO-BMSC (traceable for 30 d) and Fth1-BMSC groups. T2WI was not sensitive enough to trace Fth1-BMSCs. After transplantation, Prussian blue-stained cells were observed around the infarction area and in the infarction center in both transplantation models. Fth1-BMSCs transplanted for treating focal cerebral infarction were safe, reliable, and traceable by MRI. Fth1 labeling was more stable and suitable than SPIO labeling for long-term tracking. SWI was more sensitive than T2W1 and suitable as the optimal MRI-tracking sequence.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

Hematopoietic Repopulation, In Vivo, with Genetically Defined Clones Derived from HOXB4 Expressing ES-Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 196-196
Author(s):  
Sandra Pilat ◽  
Sebastian Carotta ◽  
Bernhard Schiedlmeier ◽  
Kenji Kamino ◽  
Andreas Mairhofer ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Es Cells ◽  
Es Cell ◽  
Somatic Gene Therapy ◽  
Hematopoietic System ◽  
Somatic Gene

Abstract In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES) cells. After in vitro differentiation, some of their progeny cells are capable of mediating long term hematopoietic repopulation after transplantation into immunodeficient recipient mice, in vivo. This is especially efficient when the homeodomain transcription factor HOXB4 is ectopically expressed (1). We have recently shown that HOXB4-ES-cell derivatives behave similar to bone marrow cells also expressing this transcription factor ectopically, both in vitro and in vivo (2). Here we demonstrate that long term repopulation (>6 months) in Rag2(−/−)γ C(−/−) mice can be achieved with ES-cell derived hematopoietic cells (ES-HCs) obtained from single, molecularly characterized ES-clones, in which the insertion sites of the retroviral expression vector had been defined. Clones expressing HOXB4 above a certain level showed a high extent of chimerism in the bone marrow of transplanted mice (average 75%; range 45–95%, n=4) whereas ES-HC clones expressing lower levels only repopulated with very low efficiency (average 2.5% chimerism, range 1–4%, n=6 mice). These results suggest that the capability of long-term repopulation, in vivo, is highly dependent on the expression levels of HOXB4 in the transplanted clones. Only mice reconstituted with ES-HC clones expressing high amounts of HOXB4 and thus showing substantial chimerism, recapitulated the morphohistological phenotype observed in polyclonally reconstituted mice. This included the bias towards myelopoiesis, “benign” myeloid proliferation in spleen and the incompatibility of HOXB4 expression with T-cell poiesis (2). In summary, we demonstrate that repopulation of the hematopoietic system can be achieved with preselected clones of genetically manipulated stem cells in which a) the insertion site of the retroviral (gene therapy) vector has been characterized prior to transplantation and b) in which ectopic HOXB4 has to be expressed above a certain threshold level. Thus, ES cells carry the potential for performing safe somatic gene therapy when using integrating gene therapy vectors. Nevertheless, advanced cell therapy will certainly require the expression of HOXB4 in a regulated manner to avoid unwanted effects such as disturbed lineage differentiation.

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