Long-term culture of hematopoietic stem cells - validating the stromal component of the CAFC assay.

BACKGROUND: The stroma-based long-term culture is the assay of choice when a functional detection of primitive hematopoietic cells in vitro is sought. However, different stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in published frequencies of LTCIC alternative CAFC. METHODS: In order to identify the most suitable stromal source in terms of supportive capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma in secondary long-term culture set-ups. RESULTS: Seeking an approximation to the supportive capacity of human BM stroma we found the FBMD-1 cell line supplemented with G-CSF and IL-3 superior to FBMD-1 cells alone, and to M2-10B4 and Sl/Sl cells. Moreover, in co-cultures of CD34(+) cells and the FBMD-1 line, we found week 5 CAFC content highly reproducible (50.5 +/- 6.66 - 54.6 +/- 7.07/10(4) plated cells, p value > 0.95) and the assay was suitable for inter-individual comparison in a clinical setting. In fact, the week 5 CAFC results were even more reproducible than those of the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU assays). On the other hand, when extending the culture period to 8 weeks, the cobblestone area formation was best maintained by human BM stroma and the high reproducibility in CAFC enumeration in cultures supported by the FBMD-1 was lost. DISCUSSION: Among the stromal cell sources tested, the FBMD-1 line was found to be superior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.

Reduced total number of cobblestone area forming cells and in vitro stromal-cell growth in autografts from acute myeloid leukemia patients.

BACKGROUND: It is well known that ABMT in acute myeloid leukemia (AML) often results in delayed hematopoietic engraftment, but the reason behind this has not been resolved. Previous studies have largely dealt with measurements of committed myeloid progenitors as surrogate markers for hematopoiesis. METHODS: Measurements of Week 5 cobblestone area forming cells (CAFC) and stromal-cell growth in BM autografts from 14 AML patients were compared with those from 10 NHL patients. RESULTS: Grafts achieved from the AML patients contained a significantly lower total number of CAFC than those from the NHL patients. The reason for this was a lower total amount of mononuclear cells (MNC) obtained during harvest procedure (mean 0.4 x 10(8)/kg for AML, versus 0.8 x 10(8)/kg for NHL). In contrast, the frequency of CAFC was comparable both between patient groups (mean 1.47, range 0.15-6.33 per 10(4) MNC for AML versus mean 1.47, range 0.53-3.57 per 10(4) MNC for NHL) and compared with that of eight normal donors (mean 1.12, range 0.73-1.73 per 10(4) MNC). An inverse relationship was observed between the total CAFC number in the grafts and the hematopoietic reconstitution of both granulocytes > or = 2.0 x 10(9)/L and thrombocytes > or = 50 x 10(9)/L, in which the level of 9.0 x 10(3) CAFC/kg implied a prompt engraftment for both patient groups. Whereas the stromal cell outgrowth in vitro from 8/10 NHL patients was similar to that of six normal donors, only a few stromal cells appeared in the majority of nine evaluable AML patients. DISCUSSION: A decreased total CAFC content, as well as an inferior stromal-cell function, may be critical elements for prolonged hematopoietic reconstitution in AML.

Histological changes in the penis of the Chacma baboon--a model to study aging penile vascular impotence.

Light and scanning electron microscopy was performed on the penes of Chacma baboons to establish their suitability as animal model for studying vascular impotence. Analogous aging penile vascular changes were found in man, and a fibrous trabeculopathy with intertrabecular senechiae was demonstrated. Considering the corresponding changes, the Chacma baboon could thus be regarded suitable for studying the pathophysiology of aging penile vascular changes.

Abnormal vessels in the urinary tract following analgesic abuse in man.

A brownish discoloration of the bladder mucosa around the ureteric orifices has been observed on cystoscopy in three patients taking excessive quantities of analgesics. Biopsy specimens revealed an unusual degree of hyalinization of the vessels in the submucosa. Similar changes have been noted in the renal medulla, pelvis, and ureter of patients with classical analgesic nephritis. This vascular change may play an important part in the development of the characteristic infarct-like necrosis seen in the analgesic kidney.

Analgesic cystitis: an unusual microangiopathy of the bladder.

A brownish discoloration of the bladder mucosa around the ureteral orifices has been observed on cystoscopic examination in 3 patients taking excessive quantities of analgesics. Biopsy revealed an unusual degree of hyalinization of the capillary walls in the submucosa.