scholarly journals Ex vivo culture of Fancc-/- stem/progenitor cells predisposes cells to undergo apoptosis, and surviving stem/progenitor cells display cytogenetic abnormalities and an increased risk of malignancy

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3465-3471 ◽  
Author(s):  
Xiaxin Li ◽  
Michelle M. Le Beau ◽  
Samantha Ciccone ◽  
Feng-Chun Yang ◽  
Brian Freie ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Genome Stability ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Acquired Resistance ◽  
Intrinsic Defects ◽  
Cytogenetic Abnormalities ◽  
Increased Risk ◽  
Ex Vivo Culture ◽  
The Impact

AbstractCurrent strategies for genetic therapy using Moloney retroviruses require ex vivo manipulation of hematopoietic cells to facilitate stable integration of the transgene. While many studies have evaluated the impact of ex vivo culture on normal murine and human stem/progenitor cells, the cellular consequences of ex vivo manipulation of stem cells with intrinsic defects in genome stability are incompletely understood. Here we show that ex vivo culture of Fancc-/- bone marrow cells results in a time-dependent increase in apoptosis of primitive Fancc-/- progenitor cells in conditions that promote the proliferation of wild-type stem/progenitor cells. Further, recipients reconstituted with the surviving Fancc-/- cells have a high incidence of cytogenetic abnormalities and myeloid malignancies that are associated with an acquired resistance to tumor necrosis factor α (TNF-α). Collectively, these data indicate that the intrinsic defects in the genomic stability of Fancc-/- stem/progenitor cells provide a selective pressure for cells that are resistant to apoptosis and have a propensity for the evolution to clonal hematopoiesis and malignancy. These studies could have implications for the design of genetic therapies for treatment of Fanconi anemia and potentially other genetic diseases with intrinsic defects in genome stability.

scholarly journals Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Daisuke Araki ◽  
Stefan Cordes ◽  
Fayaz Seifuddin ◽  
Luigi J. Alvarado ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Er Stress ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Adult ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
Human Hscs ◽  
Ex Vivo Culture ◽  
Cells Cultured

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

Adhesion to Fibronectin Maintains Regenerative Capacity During Ex Vivo Culture and Transduction of Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4612-4621 ◽  
Author(s):  
M.A. Dao ◽  
K. Hashino ◽  
I. Kato ◽  
J.A. Nolta
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Retroviral Vectors ◽  
Current Data ◽  
Regenerative Capacity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Ex Vivo Culture

Abstract Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.

Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 477-477
Author(s):  
Christopher B Cole ◽  
Angela M. Verdoni ◽  
David H Spencer ◽  
Timothy J. Ley
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Age-dependent visualization of neural progenitor cells within the rostral migratory stream via MRI and endogenously labeled micron-sized iron oxide particles

2018 ◽  
Author(s):  
Dorela D. Shuboni-Mulligan ◽  
Shatadru Chakravarty ◽  
Christiane L. Mallett ◽  
Alexander M. Wolf ◽  
Stacey Forton ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Progenitor Cells ◽  
Neural Progenitor Cells ◽  
Ex Vivo ◽  
Neural Progenitor ◽  
Rostral Migratory Stream ◽  
Iron Oxide Particles ◽  
The Impact ◽  
Migratory Stream

AbstractThe subventricular zone (SVZ) is one of the primary sources for rodent neural progenitor cells (NPC), however, aging greatly impacts the substructure of the region and rate of new cell birth. To determine if age impacts the rate of in vivo migration within animals, we examined the rostral migratory stream (RMS) of animals across 12 days using an established MRI technique. To visualize NPCs, we injected micron sized particles of iron oxide (MPIO) into the lateral ventricle to endogenously label cells within the SVZ, which then appeared as hypo-intensive spots within MR images. Our in vivo MRI data showed that the rate of migration was significantly different between all ages examined, with decreases in the distance traveled as age progressed. The total number of iron oxide labeled cells within the olfactory bulb on day 12, decrease significantly when compared across ages in ex vivo high-resolution scans. We also, for the first time, demonstrated the endogenous labeling of cells within the dentate gyrus (DG) of hippocampus. Here too, there was a significant decrease in the number of labeled cells within the structure across age. Histology of the NPCs verified the decrease in labeling of cells with doublecortin (DCX) as age progressed for both regions. The dramatic reduction of labeling in NPCs within the SVZ and DG observed with MRI, demonstrates the importance of understanding the impact of age on the relationship of NPC and disease.

scholarly journals Cytogenetic Testing by Fluorescence in Situ Hybridization Is Improved by Plasma Cell Sorting in Multiple Myeloma

2021 ◽  
Author(s):  
Jihye Ha ◽  
Hyunsoo Cho ◽  
Taek Gyu Lee ◽  
Saeam Shin ◽  
Haerim Chung ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Cell Sorting ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Poor Growth ◽  
Cytogenetic Abnormalities ◽  
Cytogenetic Testing

Abstract Accurate detection of cytogenetic abnormalities has become more important for improving risk-adapted treatment strategies in multiple myeloma (MM). However, precise cytogenetic testing by fluorescence in situ hybridization (FISH) is challenged by the dilution effect of bone marrow specimens and poor growth of plasma cells ex vivo. To address these issues, we compared the performances of three different enrichment modalities for FISH: direct FISH, fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) technique, and a plasma cell sorting FISH with fluorescence-activated cell sorter (FACS). We examined cytogenetic abnormalities in bone marrow cells of 493 patients with newly diagnosed MM and compared the efficacy of each modality. FISH disclosed cytogenetic abnormalities in 38.0% of samples by direct FISH, 56.3% by FICTION, and 95.5% by FACS-FISH, and the percentage of cells with abnormal signals detected by FISH was higher by FACS-FISH than direct FISH or FICTION. Our results suggest that the efficacy of FISH is dependent on the plasma cell enrichment modalities and reveal that plasma cell sorting FISH with FACS enables better detection of cytogenetic abnormalities in diagnostic MM samples with low plasma cell frequency.

scholarly journals Gonadotropin-Releasing Hormone in Regulation of Thymic Development in Rats: Profile of Thymic Cytokines

2019 ◽  
Vol 20 (16) ◽  
pp. 4033 ◽  
Author(s):  
Victoria I. Melnikova ◽  
Nadezhda V. Lifantseva ◽  
Svetlana N. Voronova ◽  
Liudmila A. Zakharova
Keyword(s):  
Ex Vivo ◽  
Interferon Γ ◽  
Gonadotropin Releasing Hormone ◽  
Recent Experimental Data ◽  
Mrna Levels ◽  
Fetal Thymus ◽  
Releasing Hormone ◽  
Thymus Development ◽  
Ex Vivo Culture ◽  
The Impact

An increasing body of recent experimental data confirms the impact of neurohormones on fetal development and function of different body systems. The synthesis of many neurohormones starts in fetal tissues before the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal systems are formed, and their high levels are detected in the bloodstream. Here, we studied the role of gonadotropin-releasing hormone (GnRH) in rat thymus development and tried to reveal possible mechanisms underlying the GnRH effects in early development. Western blotting and reverse transcription-polymerase chain reaction allowed us to identify receptor for GnRH in the fetal thymus with peak expression on embryonic days 17–18 (ED17–18). Blocking the receptors in utero on ED17 by a GnRH antagonist suppressed the concanavalin A-induced proliferative response of T cells in adults. GnRH (10−7 M) increased mRNA expression of interleukin (IL)-4, IL-10, IL-1β, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) in the thymus of 18-day fetuses after an ex vivo culture for 24 h. The increased mRNA levels of the cytokines in the thymus were accompanied by increased numbers of CD4+ T helpers. Overall, the data obtained confirm the regulatory or morphogenetic effect of GnRH on fetal thymus development mediated by synthesis of thymic cytokines.

scholarly journals Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
André Branco ◽  
Sara Bucar ◽  
Jorge Moura-Sampaio ◽  
Carla Lilaia ◽  
Joaquim M. S. Cabral ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Culture System ◽  
Ex Vivo ◽  
Fold Increase ◽  
Alternative Source ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Free System ◽  
Colony Forming Unit ◽  
The Impact

Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

Leukemic Predisposition of Mice Transplanted With Gene-Modified Hematopoietic Precursors Expressing flt3 Ligand

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2003-2011 ◽  
Author(s):  
Teresa S. Hawley ◽  
Andrew Z.C. Fong ◽  
Henrik Griesser ◽  
Stewart D. Lyman ◽  
Robert G. Hawley
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Transmembrane Protein ◽  
Macrocytic Anemia ◽  
Leukemic Cell ◽  
Cell Types ◽  
Soluble Form ◽  
Hematopoietic Stem

Abstract flt3/flk-2 ligand (FL) is a cytokine that exhibits synergistic activities in combination with other early acting factors on subpopulations of hematopoietic stem/progenitor cells. In addition to normal hematopoietic precursors, expression of the FL receptor, flt3R, has been frequently demonstrated on the blast cells from patients with acute B-lineage lymphoblastic, myeloid, and biphenotypic (also known as hybrid or mixed) leukemias. Because many of these leukemic cell types express FL, the possibility has been raised that altered regulation of FL-mediated signaling might contribute to malignant transformation or expansion of the leukemic clone. In humans, FL is predominantly synthesized as a transmembrane protein that must undergo proteolytic cleavage to generate a soluble form. To investigate the consequences of constitutively expressing the analogous murine FL isoform in murine hematopoietic stem/progenitor cells, lethally irradiated syngeneic mice (18 total) were engrafted with post–5-fluorouracil–treated bone marrow cells transduced ex vivo with a recombinant retroviral vector (MSCV-FL) encoding murine transmembrane FL. Compared with control mice (8 total), MSCV-FL mice presented with a mild macrocytic anemia but were otherwise healthy for more than 5 months posttransplant (until 22 weeks). Subsequently, all primary MSCV-FL recipients observed for up to 1 year plus 83% (20 of 24) of secondary MSCV-FL animals that had received bone marrow from asymptomatic primary hosts reconstituted for 4 to 5 months developed transplantable hematologic malignancies (with mean latency periods of 30 and 23 weeks, respectively). Phenotypic and molecular analyses indicated that the tumor cells expressed flt3R and displayed B-cell and/or myeloid markers. These data, establishing that dysregulated expression of FL in primitive hematopoietic cells predisposes flt3R+ precursors to leukemic transformation, underscore a potential role of this cytokine/receptor combination in certain human leukemias. © 1998 by The American Society of Hematology.

Abstract 577: Origin and Therapeutic Efficacy of Human Cardiac Progenitor Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Federico Mosna ◽  
Claudia Bearzi ◽  
Marcello Rota ◽  
Toru Hosoda ◽  
Jochen Tillmanns ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Connexin 43 ◽  
Human Heart ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Functional Integration ◽  
Phenotypic Properties ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice

To establish whether progenitor cells are present in the human heart (hCPCs) and have phenotypic properties distinct from human hematopoietic stem cells (hHSCs), samples of human myocardium were enzymatically dissociated and c-kit-positive-cells were sorted and plated immediately to obtain clones derived from single founder cells. By FACS analysis, freshly isolated hCPCs comprised 1.1±1.0% of the population and were negative for HSC markers, CD34 and CD133, and KDR. They were also negative for epitopes of monocytes, CD14 and CD16, mast cells, CD45, and lymphocytes, CD3 and CD20. The phenotype of hCPCs was completely different from that of human bone marrow cells which were positive for these surface antigens. Only small fractions of hCPCs expressed GATA4 and Nkx2.5. Of 1,530 seeded hCPCs, 11 clones were generated accounting for 0.7% cloning efficiency. Subsequently, these cells were injected in immunodeficient mice and rats at the time of coronary occlusion and 5 days after infarction. This was done to assess whether hCPCs differentiated into myocytes and coronary vessels immediately after ischemic injury and in the presence of a well-developed infarct. In both cases, hCPCs regenerated the infarcted myocardium. Connexin 43 and N-cadherin were detected between recipient rodent myocytes and newly formed human myocytes. The problem was then whether the structural integration of these two myocyte populations had a physiological counterpart. Studies were performed using an ex vivo preparation together with two-photon microscopy and laser line-scan imaging. EGFP-positive-hCPCs were injected in infarcted mice and the heart was studied 2-weeks later. The heart was perfused with an oxygenated Tyrode solution containing the calcium indicator Rhod-2 and stimulated at 1 Hz. Calcium transients was recorded in EGFP positive human myocytes and EGFP negative mouse myocytes. The synchronicity in calcium tracings between these two distinct cell pools was apparent, pointing to the functional integration of newly formed EGFP-positive human myocytes with the surrounding EGFP-negative mouse myocytes. Thus, the human heart contains hCPCs which are not of bone marrow origin and possess the ability to acquire the cardiomyocyte and coronary vascular cell lineages.

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