Towards the goal of exploiting hES cells for novel hematopoietic cell reconstitution and HIV gene therapies, here we have shown that phenotypically normal and functionally competent dendritic cells could be differentiated from hES-CD34+ cells. Moreover, we also have demonstrated for the first time that hES-DCs can be productively infected with HIV-1 thus allowing future testing of anti-HIV therapeutic genes such as siRNAs for efficacy in these cells.
In these studies, we induced hES derived CD34+ cells to differentiate into myeloid DCs in the presence of cytokines SCF, GM-CSF, Flt3, IL-3, TNF-α, and IL-4. CD34+ cells derived from human fetal liver were also evaluated in parallel for comparison. Based on FACS analysis for surface markers during culture, CD34+ cells differentiated into mature myeloid DCs showing the typical CD1a phenotype similar to those derived from FL CD34+ cells. The morphology and phenotypic characteristics of hES-DCs were found to be similar to that of DCs derived from fetal liver CD34+ cells cultured in parallel.
It is important that hES-DCs are also functionally normal for future applications. Therefore we analyzed the functional markers HLA-DR (MHC-II), B7.1 (CD80), and B7.2 (CD86) typically expressed by mature DCs. The antigen presenting cell surface marker, HLA-DR present on mature DCs is critical for antigen presentation to CD4+
T cells and the co-stimulatory molecules B7.1 and B7.2 are needed to activate T cells. The mature hES-DCs expressed HLA-DR, B7.1, and B7.2 surface molecules which were comparable with levels expressed in FL-DCs. Consistent with the DCs' ability, the hES-DCs also showed normal capacity for antigen capture as evidenced by dextran uptake. We further analyzed the capacity of hES-DCs to induce proliferation of allogeneic T cells in a mixed leukocyte reaction. Our results showed that hES-DCs indeed are capable of mediating this allogeneic response. We also evaluated the expression of another cell surface molecule, DC-SIGN (CD209) which is a DC-specific adhesion receptor belonging to the C-type lectin family involved in the interactions with T cells [13
]. Our results showed similar levels of DC-SIGN in hES-DCs and FL-DCs. The above data taken together showed that hES-DCs are phenotypically and functionally normal.
It is known that HIV-1 can infect DCs with the virus remaining stable for long periods. DCs transmit the virus efficiently to CD4 T cells and therefore play an important role in HIV-1 infection. Our results showed that hES-DCs were susceptible to HIV-1 infection similar to that of FL-DCs. However, not all the cells in the culture were productively infected and the levels of viral production are low. This is consistent with previous findings that DCs support only a low level replication and fully mature DCs may have a block in viral replication [13
]. A recent report has also demonstrated the derivation of physiologically normal DCs from hES-CD34+ cells using a different protocol and compared these to those derived from adult human CD34+ cells and peripheral blood monocytes [19
]. Our results are in agreement and confirmed these previous findings, and additionally extended them further by demonstrating that hES-DCs are also susceptible to HIV-1 infection.
In HIV-1 disease, infection of CD4 T cells leads to their eventual decline whereas infection of monocytes/macrophages and dendritic cells leads to continued viral spread and defects in antigenic presentation thus exacerbating the disease process [14
]. We previously demonstrated the derivation of macrophagres from hES-CD34+ cells whereas studies of Galic et al [20
] derived functional T cells from hES-CD34+ cells in vivo using humanized mice. Whether the hES derived T cells support HIV-1 infection remains to be determined. Lentiviral vector transduction of hES cells and derivation of functional macrophages and T cells that retained the expression of the transgene established that hES cells are tractable for deriving gene modified end-stage primary hematopoietic cells [10
]. Moreover, our present results together with our previous findings that both hES derived DCs and macrophages are susceptible to HIV infection paved the way for testing anti-HIV constructs introduced into either hES cells or their derivative hematopoietic progenitor CD34+ cells. Thus far many previous studies including our own evaluated a variety of anti-HIV gene constructs in a hematopoietic stem cell setting using CD34+ cells from routine sources such as bone marrow and cord blood [11
]. Newer and more potent novel constructs such as siRNAs are currently being investigated some of which are currently entering clinical trials [21
]. In addition to anti-HIV genes with a direct inhibitory action on viral molecules, siRNAs and ribozymes that down regulate cellular molecules that aid in HIV-1 infection such as viral coreceptors CCR5 and CXCR4 also show considerable promise [22
]. Such constructs can now be introduced into hES cells and their efficacy tested in end-stage cells represented by DCs, macrophages, and T cells.
In summary, our data demonstrated the development of terminally differentiated DCs derived from hES cells. The hES-DCs display typical DC morphology, express normal phenotypical markers, are capable of antigenic stimulation, and support HIV-1 infection.