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Heart infarct in NOD-SCID mice: Therapeutic vasculogenesis by transplantation of human CD34⁺ cells and low dose CD34⁺KDR⁺ cells

ROSANNA BOTTA^*,, ERHE GAO, GIORGIO STASSI, DESIRÉE BONCI, ELVIRA PELOSI, DONNA ZWAS^||, MARIELLA PATTI, LUCREZIA COLONNA^*, MARTA BAIOCCHI, SIMONA COPPOLA, XIN MA, GIANLUIGI CONDORELLI^*,¶,# and CESARE PESCHLE^*,,1

^* Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA;
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanità, Rome, Italy;
Center for Translational Medicine - Department of Medicine, Thomas Jefferson University, Philadelphia Pennsylvania, USA;
Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy;
^|| Department of Medicine, Division of Cardiology, Thomas Jefferson University, Philadelphia Pennsylvania, USA;
^¶ Laboratory of Molecular Cardiology, San Raffaele Science Park of Rome, Rome, Italy; and
^# Institute of Molecular Medicine, University of California, San Diego, La Jolla California, USA

¹Correspondence: Thomas Jefferson University, Kimmel Cancer Center, Bluemle Life Sciences Bldg., Room #609, 233 South 10^th St., Philadelphia, PA 19107-5541, USA. E-mail: cesare.peschle{at}mail.jci.tju.edu

SPECIFIC AIMS

Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast. Specifically, the human cord blood (CB) CD34⁺KDR⁺ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. In particular, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established.

In our studies we induced myocardial infarct (MI) in the immunocompromised NOD-SCID mouse model and monitored therapeutic effects of myocardial transplantation of human CB CD34⁺ cells, as well as of CD34⁺KDR⁺ and CD34⁺KDR^– subfractions (Fig. 1 ). The specific aims of our studies were to 1) determine the effect of unseparated CB CD34⁺ cells vs. PBS and mononuclear cells (MNCs); 2) compare the action of the CD34⁺KDR⁺ cell subfraction vs. the CD34⁺KDR^– subset; and 3) conduct coculture experiments in order to assess in vitro cardiomyogenic potential of CD34⁺ and CD34⁺KDR⁺ cells as compared with the CD34⁺KDR^– cell subset.

View larger version (20K): [in this window] [in a new window]   Figure 1. Extension of the infarct area in NOD-SCID mice injected with unseparated CD34+ or CD34+KDR+ cells as compared to PBS or CD34+KDR– injected mice 3 wk after MI. A) Representative TTC staining in hearts from mice transplanted with CD34+, CD34+KDR+, or CD34+KDR– cells or injected with PBS after MI, as compared to Sham control. B) Size of infarct area at 21 days after MI in mice injected with CD34+ cells (n=6), CD34+KDR+ or CD34+KDR– cells (n=4), PBS (n=8) or sham control mouse. Infarction area is expressed as percentage of necrotic tissue by TTC staining. Mean + SEM values are presented. **P<0.01 vs. PBS and KDR– group.

PRINCIPAL FINDINGS

1. Resistance of CD34⁺KDR⁺ cells to apoptosis in serum-free culture via autocrine VEGF release.
In vitro assays demonstrated that a low number of CD34⁺KDR⁺ cells release a significant amount of autocrine VEGF, which renders cells resistant to apoptosis even in serum-free starvation culture. CD34⁺KDR^– cells, seeded at the same density and in equivalent culture conditions, release a markedly lower amount of VEGF and rapidly undergo apoptosis.

2. Transplantation of CB CD34⁺ cells improves left ventricular (LV) function (Fig. 2) .
Hemodynamic assessment by Millar catheterization demonstrated a significant increase in +dP/dt (a key index of systolic function) and -dP/dt (a key index of diastolic function), as well as a significant drop of LV end diastolic pressure (LVEDP) 21 days after MI in mice transplanted with 2x10⁵ CD34⁺ cells, as compared to mice transplanted with PBS solution. This effect is maintained 5 months after MI.

View larger version (19K): [in this window] [in a new window]   Figure 2. Hemodynamic effects of CB cell injection 3 wk after MI in NOD-SCID mice. Hemodynamic parameters in mice injected with CD34+ cells and MNCs (2x105cells), CD34+KDR+ and CD34+KDR– (2x103cells) cells after MI, as compared to Sham and PBS control groups. Analysis was performed 21 days after induction of myocardial infarction and cell transplantation. Mean + SE data from 4–9 animals per group are presented. Upper panel: Left ventricular end diastolic pressure (LVEDP) evaluation. Hearts injected with CD34+KDR+ cells showed significantly lower values than those injected with PBS or CD34+KDR– cells (°P<0.05) as did CD34+ vs. PBS-injected hearts(**P<0.01); N.D., not done. Middle panel: Evaluation of the derivative of pressure over time (+dP/dt). CD34+ and CD34+KDR+ groups showed significant higher values, as compared to PBS control and CD34+KDR– groups (*P<0.05 for CD34+ vs. PBS; °°P <0.01 for CD34+KDR+ vs. PBS or CD34+KDR–). Lower panel: Evaluation of (–)dP/dt showed higher values for CD34+ group as compared to PBS control (*P<0.05) and for CD34+KDR+ cells vs. PBS or CD34+KDR– groups (°°P<0.01).

3. Animals transplanted with 2x10³ CD34⁺KDR⁺ cells also showed a significant improvement of +dP/dt and -dP/dt, as compared to those transplanted with the same number of CD34⁺KDR^– cells (Fig. 2) .

4. Infarct size, evaluated by TTC analysis at 21 days after MI, shows a highly significant reduction in CD34⁺ and CD34⁺KDR⁺ transplanted mice, as compared to PBS and CD34⁺KDR^– transplanted controls (Fig. 3) .
Moreover, in CD34⁺ and CD34⁺KDR⁺ transplanted mice a decrease of both CMC apoptosis and fibrosis were observed.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram. Myocardial infarct (MI) was induced in the immunocompromised NOD-SCID mouse model by ligation of left anterior descending coronary artery (LAD). Cells (2x105 CD34+ or MNCs; 2x103 FACS sorted CD34+KDR+ or CD34+KDR–) were then injected into the peri-ischemic area. After 21 days, mice were studied for hemodynamic parameters, infarct size was quantified by TTC staining, and histological analysis was performed on myocardial tissue in order to investigate generation of newly formed cells by human CD34+ and CD34+KDR+ cells transplanted in the peri-infarct area.

5. In vitro coculture assays indicate that CD34⁺ and CD34⁺KDR⁺ cells are able to convert into CMCs and a cell fusion mechanism cannot be excluded.

CONCLUSIONS AND SIGNIFICANCE

We describe here the beneficial effects on cardiac function of human CB CD34⁺ cells transplanted after MI in the NOD-SCID mouse model. This effect is demonstrated by invasive hemodynamic techniques. Moreover, we show that a small subfraction of these cells, the CD34⁺KDR⁺ subset, is endowed with a marked therapeutic potential, since its beneficial effect on hemodynamic parameters compares with that observed after transplantation of a two log higher number of CD34⁺ cells. Because the CD34⁺KDR⁺ subset constitutes 1% of CD34⁺ cells, results suggest that CD34⁺KDR⁺ cells represent the therapeutically active subfraction within CD34⁺ cell population. Accordingly, the CD34⁺KDR^– subset exerts little or no beneficial effect.

We show here that human CD34⁺ cells are able to convert into CMCs in vivo, although at a limited efficiency. Due to the low number of HNA⁺ nuclei within a CMC context, we cannot rule out the possibility that the few CMCs newly formed in the transplanted heart are generated by cell fusion. This mechanism cannot be excluded in our in vitro coculture assays, which suggest that CMCs may be generated from both CD34⁺ and CD34⁺KDR⁺ cells.

Our results showed the number of CMCs newly formed in vivo from CD34⁺ cells to be at least two log lower than previously reported in a syngeneic murine model transplanted with primitive hematopoietic cells. In human transplanted hearts, it has been demonstrated that CMCs can be generated from bone marrow (BM) stem cells, although conflicting data exist on the extent of this phenomenon. In subsequent reports, the number of newly formed CMCs from BM stem cells in transplanted patients was much lower. Similarly, the BM cell subset isolated through Hoechst staining uptake ("side-population") and transplanted into irradiated NOD-SCID mice was endowed with neocardiomyogenic potential, but only at a limited extent. In line with these findings, results presented here suggest that CD34⁺KDR⁺ Hem-End primitive cells have cardiomyogenic potential at a limited extent.

We previously showed that End progenitor cells with hematopoietic potential from the murine embryonic dorsal aorta, as well as HUVEC cells, are able to differentiate in CMCs in coculture experiments. More recently, it has been shown that human End progenitor cells from peripheral blood (PB) were also able to differentiate into CMCs in coculture assays. In our studies, the fraction of CB CD34⁺ cells differentiating toward angiogenesis and cardiomyogenesis is apparently represented by the CD34⁺KDR⁺ subfraction. This cell subset, enriched for End progenitors and hemangioblasts, might be related to End precursor populations reportedly endowed with cardiomyogenic potential.

CD34⁺ and CD34⁺KDR⁺ CB cells, while of limited cardiomyogenic potential, have a marked angiogenetic capacity, providing hope for a therapeutic use of these cells in chronic ischemic disorders. Angiogenic potential is in fact elevated, as shown by the low level of both fibrosis and CMC apoptosis in the myocardium of mice receiving an injection of CD34⁺ or CD34⁺KDR⁺ cells. Similarly, a decrease in cardiomyocyte apoptosis linked to an improvement of cardiac function was obtained by injection of human peripheral blood CD34⁺ cells in the tail vein of immunocompromised rats after MI. Unlike previously reported results which utilized a large number of MNCs or End cells ex vivo expanded from BM MNCs, our model, using a low number of MNCs, was not able to induce angiogenesis and improve heart function.

Therapeutic efficacy of a small number of CD34⁺KDR⁺ cells is a novel and significant finding, seemingly mediated by diverse factors. 1) As mentioned above, the CD34⁺KDR⁺ subfraction is not only enriched for Hem and End progenitor/stem cells, but also comprises hemangioblasts (i.e., cells with capacity to generate both Hem and End progeny). Single cell culture studies indicated that these functionally different cell populations may represent a single cell pool exhibiting hematopoietic and/or endothelial differentiation in different microenvironmental conditions. In the MI model utilized here, transplanted CD34⁺KDR⁺ cells may be channeled into End differentiation, and limited CMC generation. 2) CD34⁺KDR⁺ cells are highly resistant to apoptosis in serum-free GF-starved culture via autocrine VEGF release. Conversely, CD34⁺KDR^– cells rapidly die in these harsh culture conditions. We suggest that CD34⁺KDR⁺ (but not KDR^–) cells may be particularly resistant to apoptosis when transplanted in the heart infarct area. Ongoing studies aim to verify this aspect. 3) A small number of transplanted CD34⁺KDR⁺ cells releasing significant amounts of VEGF may exert an anti-apoptotic and proliferative action on murine heart endothelial cells, thus enhancing capillarization and improving survival of CMCs. Hypothetically, CD34⁺KDR⁺ cells may activate cardiac stem cells and/or release factors exerting a direct antiapoptotic effect on CMCs. Preliminary coculture studies suggest in fact a direct antiapoptotic action.

Our results indicate that, within CD34⁺ cell population, the CD34⁺KDR⁺ fraction is responsible for improvement in cardiac hemodynamics and hence represents the candidate active CD34⁺ cell subset. Admittedly, the number of available CD34⁺KDR⁺ cells is limited. In this regard, ongoing studies in our laboratory aim to expand ex vivo the CD34⁺KDR⁺ subset, in order to facilitate its therapeutic use at preclinical and then possibly clinical level.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0879fje;

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