Adult marrow cells cocultured with murine aft024 and defined cytokines give rise to nk cells, b cells, dendritic cells and myeloid cells from the same single sorted multi-lineage progenitor

Human CD34+/Lin-/DR- cells develop into NK celt progeny after longterm culture in contact with primary altogeneic stroma and IL-2 but B cells never develop. We tested whether different microenvironments could induce not only NK cells but also B cells from these lineages that depend on a marrow microenvironment for differentiation. CD34+/Lin-/DR- cells were plated in limiting dilutions for 3-5 weeks with IL-7, flt3 ligand (FL), c-kit ligand (KL), and IL-2 in absence or present of two murine stromal lines: M2-10B4 which supports myeloid progenitors and AFT024. a murine fetal liver line recently described to support culture of transplantable stem cells from murine marrow. Culture without a feeder layer failed to result in viable cell progeny. Culture in the presence of M2-10B4 resulted in phenotypic CD56+/CD3- NK cells at low frequency but without B cells. In contrast, in the AFT024 cultures, both NK cells and CD10+/CDI9+ B cells could be found in the same wells. B cells were immature based on the absence of surface heavy or light chains and the presence of TdT. B cell differentiation was dependent on exogenous IL-7 and FL added to cultures. The role of IL-7, FL, KL, IL-2 and IL-3 on cloning frequency and proliferation of NK cells, B cells and dendritic cells was established from limiting dilution assays using different cytokine combinations. Single cell deposition of 3,872 CD34+/Lin-/CD38- cells onto AFT024 further defined this in vitro model of multilineage differentiation. At the single cell level, the one time addition of 5 ng/ml IL-3 at culture initiation was essential for NK cell, B cell and dendritic cell differentiation and proliferation. Single CD34+/Lin-/CD38- cells cultured on AFT024 with IL-7, FL, KL, IL-2 and IL-3 resulted in 18% of single cell progeny positive for NK cells, 17% positive for dendritic cells, 28% positive for myeloid cells and 2% positive for B cells. Under these same culture conditions, 2% of single cells could give rise to three lineages (NK cells, B cells and dendritic cells or myeloid cells) from the same CD34+/Lin-/CD38- cell. After 6 weeks, transfer of single cell progeny to media with IL-2 alone for addition 2 weeks resulted in an average of 8.3 x 10' NK cells which exhibited characteristic cytotoxicity against K562 targets. The AFT024 murine fetal liver cell line provides a unique, highly efficient, microenvironment to study the quality of primitive progenitors by inducing multilineage differentiation at the single cell level.