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.

scholarly journals HOXB4 Enforces Equivalent Fates of ES-Cell-Derived and Adult Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 494-494 ◽  
Author(s):  
Sandra Pilat ◽  
Carotta Sebastian ◽  
Schiedlmeier Bernhard ◽  
Modlich Ute ◽  
Kamino Kenji ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Es Cell ◽  
Somatic Gene Therapy ◽  
Adult Bone Marrow ◽  
Somatic Gene ◽  
Adult Bone

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 <i>in vitro</i> selection and clonal expansion are only available for embryonic stem (ES−) cells. Previously, it has been shown that <i>in vitro</i> differentiated ES-cells engraft when ectopically expressing HOXB4, but it remained unclear whether these cells could fully resemble adult bone marrow function after transplantation<sup>1</sup>. We here demonstrate for the first time the functional equivalence of <i>in vitro</i> differentiated ES-cells and adult bone marrow cells mediated by HOXB4. Differentiated ES-cells expressing HOXB4 from a retroviral vector and grown <i>in vitro</i> for 20 days, recapitulated the growth and differentiation properties of adult bone marrow cells after transplantation into Rag2<sup>(−/−)</sup>γC<sup>(−/−)</sup> and C57Bl/6J recipient mice. Furthermore, we show that the amount of ectopically expressed HOXB4 influences differentiation in both systems in a similar manner. HOXB4 enforced myeloid and suppressed T-lymphoid development over a wide range of expression levels, whereas only high expression levels of HOXB4 were detrimental for erythroid development (P-values for C57Bl/6J mice, Student’s t-test, 2-sided: CD3 + : eGFP<sup>low</sup> vs. HOX<sup>low</sup> = 0.003; eGFP<sup>high</sup> vs. HOX<sup>high</sup> = 0.021; Ter119 +: eGFP<sup>low</sup> vs. HOX<sup>low</sup> = 0.920; eGFP<sup>high</sup> vs. HOX<sup>high</sup> = 0.0122; HOX<sup>low</sup> vs. HOX<sup>high</sup> = 0.005). Incompatibility of high levels of HOXB4 expression with erythroid differentiation was also directly demonstrated using a recently described <i>in vitro</i> ES-cell differentiation system<sup>2</sup>. Histological analysis of the “HOXB4-transplanted” mice revealed increased granulopoiesis both in sternal bone marrow and in spleen sections. However, all stages of granulocytic differentiation were present and neither were immature cells detected in the periphery nor was leukemic infiltration detected in other organs. Hence, none of the animals became leukemic during the observation period. In summary, ES-cells should be considered a promising alternative to bone marrow stem cells carrying the potential of safe somatic gene therapy, provided that human ES-cells can be similarly manipulated. Nonetheless, advanced cell therapy will certainly require regulated expression of HOXB4 to avoid unwanted effects such as disturbed lineage differentiation. This work was supported by the German Research Foundation (KL1311/2-3 and 2-4), German Cancer Aid (10-1763-OS5) and the Austrian Industrial Research Promotion Fund (808714).

scholarly journals A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3097-3105 ◽  
Author(s):  
Brandon K. Hadland ◽  
Stacey S. Huppert ◽  
Jyotshnabala Kanungo ◽  
Yingzi Xue ◽  
Rulang Jiang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Fetal Liver ◽  
Es Cells ◽  
Es Cell ◽  
Chimeric Mice ◽  
Developmental Potential ◽  
Definitive Hematopoiesis

Abstract Notch1 is known to play a critical role in regulating fates in numerous cell types, including those of the hematopoietic lineage. Multiple defects exhibited by Notch1-deficient embryos confound the determination of Notch1 function in early hematopoietic development in vivo. To overcome this limitation, we examined the developmental potential of Notch1–/– embryonic stem (ES) cells by in vitro differentiation and by in vivo chimera analysis. Notch1 was found to affect primitive erythropoiesis differentially during ES cell differentiation and in vivo, and this result reflected an important difference in the regulation of Notch1 expression during ES cell differentiation relative to the developing mouse embryo. Notch1 was dispensable for the onset of definitive hematopoiesis both in vitro and in vivo in that Notch1–/– definitive progenitors could be detected in differentiating ES cells as well as in the yolk sac and early fetal liver of chimeric mice. Despite the fact that Notch1–/– cells can give rise to multiple types of definitive progenitors in early development, Notch1–/– cells failed to contribute to long-term definitive hematopoiesis past the early fetal liver stage in the context of a wild-type environment in chimeric mice. Thus, Notch1 is required, in a cell-autonomous manner, for the establishment of long-term, definitive hematopoietic stem cells (HSCs).

CD34+ Cells Derived from Human Embryonic Stem Cells Demonstrate Hematopoietic Stem Cell Potential In Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 564-564 ◽  
Author(s):  
Dan S. Kaufman ◽  
Petter S. Woll ◽  
Colin H. Martin ◽  
Jon L. Linehan ◽  
Xinghui Tian
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Es Cells ◽  
Es Cell ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Es Cells

Abstract We have previously described methods to use either stromal cell co-culture or embryoid-body (EB) formation to support the hematopoietic development of undifferentiated human ES cells (both H1 and H9 cell lines). Either FBS-based media or serum-free media with specific cytokines can be used to derive CD34+ cells, CD45+ cells and hematopoietic progenitors as identified by colony-forming cell (CFC) assays that give rise to mature myeloid, erythroid, and megakaryocytic cells. Genes such as RUNX1, HOXB4, TAL1, and GATA2, all known to be expressed during early hematopoiesis are up-regulated during hematopoietic differentiation of human ES cells. Here, we advance these studies to demonstrate that human ES cell-derived CD34+ cells function as early hematopoietic precursors in surrogate hematopoietic stem cell (HSC) assays. The long-term culture initiating cell (LTC-IC) assay is commonly used to quantify hematopoietic precursors that can be maintained in culture for 5 or more weeks. Human cord blood (CB)-derived CD34+ cells have a LTC-IC frequency of approximately 1:30. We demonstrate LTC-ICs can also be identified from human ES cell-derived CD34+ at a frequency of approximately 1:400. These results suggest CD34+ cells from human ES cells are more heterogeneous than CD34+ cells from CB. Furthermore, we now demonstrate in vitro culture of human ES cell-derived CD34+ cells identify these cells as lymphocyte precursors. Here, we used a natural killer (NK) cell-initiating cell assay (NK-IC) where CD34+ cells are cultured on AFT024 stromal cells in media containing IL15, IL7, and other defined cytokines for 2–4 weeks. Under these conditions, both CB and human ES cell-derived cells give rise to lymphoid cells (NK cells) with over 40% CD45+CD56+ cells. Under alternative culture conditions, CD3+ T cells can also be produced from CD34+ human ES cell-derived cells. Therefore, CD34+ cells derived from human ES cells represent both myeloid and lymphoid precursor cells. Since it is not possible to define a HSC population based solely on in vitro assays, we have examined the potential for human ES cell-derived hematopoietic cells to engraft in sublethally irradiated NOD/SCID mice. Detection of scid-repopulating cells (SRCs) are considered a better surrogate for HSCs. Bone marrow, peripheral blood, and splenocytes were examined for human CD34+ and CD45+ cells 3–6 months after injection of human ES cell-derived blood cells. PCR for human chromosome 17-specific alpha-satellite DNA was also done to confirm the presence of human cells in all mice showing evidence of engraftment. We consistently find stable engraftment with 0.5–3% human CD45+ cells in the bone marrow of these mice. To better define these cells as HSCs, secondary transplants also demonstrate stable engraftment. Importantly, no teratomas are demonstrated in mice injected with differentiated human ES cells. These results demonstrate that HSCs with long-term engraftment and multi-lineage potential can be routinely and efficiently generated from human ES cells.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Treatment of Inherited Neurometabolic Diseases: The Future

1992 ◽  
Vol 7 (1_suppl) ◽  
pp. S132-S140 ◽  
Author(s):  
Pinar T. Ozand ◽  
Generoso G. Gascon
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Autologous Bone Marrow ◽  
Marrow Transplant ◽  
Somatic Gene Therapy ◽  
Autologous Bone Marrow Transplant ◽  
Somatic Gene ◽  
Dividing Cells ◽  
Normal Gene ◽  
Religious Considerations

The past 10 years' experience with bone marrow transplantation from normal, immunologically compatible donors indicates its possible use in various neurometabolic diseases, particularly in a patient who has not suffered irreparable brain damage. This experience may be a prelude to treatment by somatic gene therapy. This can be applied as an autologous bone marrow transplant, grafting the patient's own stem cells inserted with the normal gene. Although somatic gene therapy will be relatively easy for tissues with dividing cells, its application to target tissues with little or no cell division may pose difficulties. Meanwhile, techniques for the preservation, culture, and grafting of fetal neurons in humans have been developed and have been used in the treatment of Parkinson's disease. These procedures could readily be transferred to the treatment of other neurodegenerative diseases that cause significant morbidity, but ethical, legal, and religious considerations must be taken into account. All these efforts promise novel and improved management of inborn neurometabolic errors. (J Child Neurol 1992;7(Suppl):S132-S140.)

Abstract 229: The Novel Angiogenic Cytokine Secretoneurin promotes Angiogenesis, Arteriogenesis and Vasculogenesis in the Hind-Limb Ischemia Model

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Wilfried Schgoer ◽  
Margot Egger ◽  
Arno Peer ◽  
Johannes Jeschke ◽  
Ivan Tancevski ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Green Fluorescence Protein ◽  
Limb Ischemia ◽  
Promoter Sequence ◽  
The Novel ◽  
Gene Therapy Vector ◽  
Adductor Muscles

Introduction - Secretoneurin (SN) represents a sensory, inflammatory neuropeptide which was recently demonstrated to act as an angiogenic and vasculogenic cytokine in vitro and in vivo. The present study was conducted to test the hypothesis that SN may be implicated in reparative angiogenesis. Furthermore, we challenged the healing potential of SN applied as a newly generated SN gene therapy vector in the setting of limb ischemia. Methods and Results - We cloned the human SN coding sequence into the pAAV plasmid containing a cytomegalovirus enhancer/promoter sequence. Bioactivity of recombinant SN was shown by proliferative and chemotactic activity on endothelial cells in vitro. Unilateral limb ischemia was induced in C57/bl mice by femoral artery resection. By Real Time PCR, Western Blotting, SN-specific RIA and Immunhistochemistry, we documented that SN is up-regulated in ischemic muscles. Next, we tested whether SN gene therapy may exert curative effects in this ischemia model. Injection of the SN plasmid into ischemic adductor muscles increased capillary (0.67 vs. 0.35, n = 24, p = 0.02) and arteriole (0.16 vs. 0.8, n = 24, p = 0.04) density, reduced endothelial cell apoptosis, and accelerated perfusion recovery as shown by Laser Doppler Perfusion Index (LDPI ratio ischemic/control leg after 28 days of ischemia 1.1 vs. 0.7, n = 24, p < 0.01) in comparson to pAAV-GFP (green-fluorescence protein) treated mice. Furthermore, SN gene therapy significantly reduced toe necrosis of ischemic limbs compared to control animals (26% vs. 50%, n = 24, p < 0.05). In bone marrow transplantation models, increased vascularity of ischemic hind-limbs after SN gene therapy was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells. Conclusions -These results suggest that the novel angiogenic cytokine Secretoneurin is up-regulated by ischemia in skeletal muscle cells. Furthermore, results from gene therapy in this ischemia model suggest that Secretoneurin represent a promising new substance for therapeutic angiogenesis.

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

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.

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.

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