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

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Abstract

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.

Original languageEnglish (US)
Number of pages1
JournalExperimental Hematology
Volume26
Issue number8
StatePublished - Dec 1 1998

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