[Cell culture for transfusion purposes: the case of red blood cells]. / Culture de cellules à visée transfusionnelle: le cas des globules rouges.

One has been trying for several years to find a substitute for red blood cells (RBC). The development of chemical or natural molecules to replace hemoglobin has nevertheless proved difficult and artificial blood is still unattainable. We have described a methodology permitting the massive ex vivo production of mature human RBC having all the characteristics of native adult RBC from hematopoietic stem cells (HSC) of diverse origins: blood, bone marrow or cord blood. This protocol allows both massive expansion of the HSC/progenitors and their complete differentiation to the stage of perfectly functional mature RBC. The levels of amplification obtained (10(5) to 2 x 10(5)) are compatible with an eventual transfusion application. We discuss here the state of the art of this new concept and evoke the obstacles to be overcome to pass from the laboratory model to clinical practice. This concept of "cultured RBC" opens up potentially considerable therapeutic perspectives in the field of blood transfusion.

Evidence for absence of toxicity of T101 immunotoxin on human hematopoietic progenitor cells prior to bone marrow transplantation.

T101-ricin A-chain immunotoxin is a hybrid molecule made up of the T101 monoclonal antibody bound to the A-chain of ricin. It specifically destroys cells expressing the cell surface T65 antigen. We have designed a preclinical study to evaluate its possible use for the in vitro treatment of T-cell hematological cancers prior to autologous bone marrow transplantation. The data presented here show that conditions previously defined to produce high tumor cell killing, i.e., a 20-hr incubation at 37 degrees in the presence of T101-ricin A-chain immunotoxin up to 10(-7) M in a 10 mM ammonium chloride solution, do not affect the in vitro proliferative capacity of human hematopoietic stem cells studied by means of semisolid medium cultures (granulocyte-macrophage progenitors, burst-forming units-erythrocyte) and continuous liquid cultures (pre-granulocyte-macrophage progenitors). Therefore, autologous bone marrow transplantation with T101-ricin A-chain immunotoxin-treated graft should be feasible.

The role of autologous bone marrow transplantation in 46 adult patients with non-Hodgkin's lymphomas.

Forty-six patients with non-Hodgkin's lymphoma (NHL) were treated with autologous bone marrow transplantation (ABMT) in two different institutions. All patients were pretreated with conventional chemotherapy. Three different conditioning regimens were used, and 20 patients underwent bone marrow purging. Twelve patients were treated in first complete remission (CR); eight are in unmaintained CR 8 to 104 months after ABMT. Five patients were grafted in first partial remission (PR) after conventional therapy; all achieved CR, and all remain in prolonged CR (first CR for four patients, second CR for one patient). Of 21 patients with chemosensitive relapses, 13 patients are in prolonged unmaintained CR 8 to 94 months after ABMT. Eight patients with resistant disease remained uncured by ABMT; all eight died, six from progressive illness and two from toxicity. The current 3-year disease-free probability is 60% for all patients, 0% for refractory disease; 82% for first PR or CR, and 60% for sensitive relapses (SRs). These results confirm the efficacy of ABMT in the treatment of chemosensitive NHL with bad prognosis.

Autologous stem-cell transplantation for non-Hodgkin's lymphomas: the role of graft purging and radiotherapy posttransplantation--results of a retrospective analysis on 120 patients autografted in a single institution.

PURPOSE: To analyze retrospectively survival and prognostic factors of patients with non-Hodgkin's lymphoma (NHL) autografted from 1979 to 1995 in a single institution. PATIENTS AND METHODS: A total of 120 patients, 64 with aggressive and 56 with low-grade NHL, were autografted. The carmustine (BCNU), etoposide, cytarabine, and melphalan (BEAM) regimen was used in 104. The autograft was marrow in 101 patients. Marrow was purged in vitro by mafosfamide for 63 patients (adjusted dose [AD] in 32; unique dose [UD] in 31); 27 patients received a CD34+-selected graft. Following intensification, 45 patients received additional radiotherapy on previous sites of involvement. RESULTS: Outcome at 5 years for patients transplanted with low-grade NHL in first complete remission (CR1), in first partial remission (PR1), and in second complete remission (CR2) or beyond showed an event-free survival (EFS) of 75% +/- 12%, 46% +/- 18%, and 57% +/- 24%, a relapse incidence (RI) of 21% +/- 12%, 49% +/- 19%, and 43% +/- 25%, and a transplant-related mortality (TRM) of 5% +/- 5%, 10% +/- 7%, and 0%, respectively. For patients with aggressive NHL transplanted in CR1, in PR1, in CR2 or beyond, and in resistant relapse or in primary refractory disease, the EFS was of 73% +/- 9%, 58% +/- 19%, 29% +/- 16%, and 10% +/- 9%, the RI 22% +/- 9%, 14% +/- 9%, 77% +/- 18%, and 66% +/- 20%, and the TRM 6% +/- 6%, 32% +/- 21%, 11% +/- 10%, and 71% +/- 22%, respectively. In patients autografted upfront in first remission, additional radiotherapy was associated with a higher EFS, in univariate (P = .03) and multivariate analysis (P = .02, relative risk [RR] = .021). The role of graft purging with mafosfamide on the outcome reflected by the dose of colony-forming unit-granulocyte-macrophage (CFU-GM) per kilogram infused postpurging was assessed by univariate analysis: patients in first remission who received lower doses of CFU-GM had a lower RI and a higher EFS. CONCLUSION: This retrospective analysis suggests that marrow purging and posttransplant radiotherapy improve the outcome of patients with NHL autografted in first remission.

IgH DJ rearrangements within T-ALL correlate with cCD79a expression, an immature/TCRgammadelta phenotype and absence of IL7Ralpha/CD127 expression.

cCD79a and IgH VDJ/DJ rearrangements are considered to be relatively specific for B lymphoid precursors. We looked for both in cCD3+, CD7+, CD19- T-ALLs classified by TCR status into alphabeta or gammadelta/immature (IM) lineages, with individualization of HOX11L2+ T-ALLs since they represent an intermediate alphabeta/gammadelta category. cCD79a was expressed at low levels in 47% of T-ALL and was most frequent in IMgamma T-ALLs. IgH rearrangements were common in gammadelta/IM (45%) and HOX11L2+ (35%) T-ALLs compared to HOX11L2-negative cases (3%; P<0.001). CD127 (IL7Ralpha) expression was also more common in the gammadelta/IM lineage but its expression was virtually mutually exclusive of IgH rearrangement. Low-level cCD79a expression alone should therefore not be interpreted as evidence of B lineage affiliation in immature leukemias. gammadelta/IM lineage T-ALLs potentially include two distinct categories: predominantly IgH+, cCD79a+, CD127- cases which retain gammadelta and B lymphoid potential and IgH-, cCD79a-, CD127+ cases with restricted T lineage potential.

Autologous bone marrow transplantation as consolidation therapy may prolong remission in newly diagnosed high-risk follicular lymphoma: a pilot study of 34 cases.

We evaluated early intensification followed by autologous bone marrow transplantation (ABMT) using marrow purged by mafosfamide in patients with high-risk low-grade follicular lymphoma (LGFL) reaching a status of minimal disease (MD). Thirty-four patients entered the program. All fulfilled at least one of the following criteria at diagnosis: a bulky tumor > 7 cm; three or more adenopathies > 3 cm; massive pleural or peritoneal effusion; massive splenomegaly; B symptoms; platelet count < 100 x 10(9)/l. Twenty-one patients had bone marrow involvement. Twenty-six patients received ACVBP, and eight CVP as front-line therapy. Twenty-one (62%) patients achieved MD status, 18 reached intensification. At 4 years, the time to treatment failure is 55 +/- 9%, and the probability of persisting remission is 75 +/- 11%. Comparison by intention to treat of the 26 patients who received ACVBP as front-line therapy to 14 historical high-risk LGFL similarly treated in our institution without intensification, showed better results for the intensified group (P = 0.04 for both probability of persisting remission and time to treatment failure). These results indicate that early intensification using marrow purged with mafosfamide is a therapeutic option which may bring benefit to patients with high-risk LGFL.

Multiple chromosome abnormalities in patients with acute leukemia after autologous bone marrow transplantation using total body irradiation and marrow purged with mafosfamide.

Cytogenetic follow-up studies such as those reported after allogeneic bone marrow transplantation are not available in patients submitted to an autologous bone marrow transplantation (ABMT). Of 114 patients with acute leukemia (69 acute myelocytic AML, 43 acute lymphocytic ALL, 2 undifferentiated) who underwent an ABMT in our institution in the period from February 1983 to December 1989, 66 had evaluable cytogenetic data post-transplant. They all received a pretransplant regimen consisting of cyclophosphamide (CY) and total body irradiation (TBI) followed by reinfusion of marrow purged with mafosfamide. Twenty patients showed chromosomal damage at some time; of these, six relapsed early post-ABMT, one died while in persisting remission at 81 months post-ABMT from overwhelming pneumococcal sepsis related to a previous splenectomy, and 13 are still alive and well at 13 to 88 months post-transplant. The bone marrow cytogenetic abnormalities were complex: they included various numbers of clonal aberrations or variations or combination of those; they affected all but the Y chromosome, with a predominance however for chromosomes 1, 3, 6, and 7; they were often transitory and in some instances became modified with time. None of these chromosomal abnormalities was connected with the initial leukemia, even in the 6 patients who relapsed early. In the other 14 patients, these abnormalities have so far had no detectable unfavourable implication. The origin of these abnormalities is unknown: both the pretransplant regimen (CY and/or TBI) and/or marrow purging with mafosfamide can be incriminated. Additional studies in patients autografted with pretransplant regimen not containing TBI and/or with unpurged marrow are necessary to discriminate between these two possibilities.

Autologous bone marrow transplantation with marrow purged by mafosfamide in seven patients with myelodysplastic syndromes in transformation (AML-MDS): a pilot study.

Seven patients with acute myeloblastic leukemia (AML) occurring on myelodysplastic syndromes (MDS) were consolidated while in complete remission (CR) by autologous bone marrow transplantation (ABMT) with a marrow purged in vitro by mafosfamide. The median age of population was 44 years (range 39-55). MDS FAB diagnosis was established before progression to AML in five patients: refractory anaemia with excess of blast (RAEB) in three patients, RAEB in transformation (RAEB-t) in one patient, and chronic myelomonocytic leukemia (CMML) in one patient. In the remaining two patients, the diagnosis of MDS (as a secondary malignancy in one) was made retrospectively at time of overt AML. Three out the seven patients had karyotypic abnormalities. The median interval between the obtention of CR and ABMT was 7 months (range 6-18). One patient died from transplant related toxicity. Engraftment occurred at a median of 41 days (range 27-60), for white blood cells (> 10(9)/l) and 120 days (range 60-180) for platelets (> 50 x 10(9)/l). Four patients relapsed at 2.5, 6.8, and 25 months post-ABMT. Two patients are alive and well at 10 and 28 months, respectively. ABMT with marrow purged by mafosfamide is feasible in patients with AML following MDS with a prospect of cure. However, further studies are needed to assess the real value of this approach.

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures.

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.

Establishment of a reliable experimental procedure for bone marrow purging with mafosfamide (ASTA Z 7557).

Bone marrow purging with cyclophosphamide derivatives (Mafosfamide) requires the establishment of a defined experimental procedure for reliable leukemic cell destruction while sparing normal hematopoietic stem cells to ensure engraftment. We previously defined the granulocyte-macrophage colony-forming unit (CFU-GM) LD95 as being the maximum tolerable dose of drug to use. We now report, in 20 patients with acute non-lymphoblastic leukemia (n = 5), acute lymphoblastic leukemia (n = 5), chronic myelogenous leukemia (n = 5), and non-Hodgkin's lymphoma (n = 5), that the nature of the cells treated (i.e., buffy coat cells or mononuclear cells) significantly influences the accuracy of the LD95 determination, whereas other parameters such as hematocrit or nucleated cell concentration do not. We subsequently define the most reliable experimental procedure for in vitro purging with Mafosfamide: incubation of 2 x 10(7) buffy coat cells/ml with a hematocrit of 5%. We show that the wide individual susceptibility to the drug is not related to any incubation procedure. In a series of 163 patients with hematological malignancies, we confirm the large variation of sensitivity to the drug according to patient susceptibility and diagnosis. These data favor the adjustment of the dose of Mafosfamide on an individual basis, prior to bone marrow purging for autologous bone marrow transplantation.

The role of serum lipoproteins on the in vitro proliferative potential of human hematopoietic progenitors CFUC and CFUE.

The influence of various lipoprotein fractions on the proliferation of normal human hematopoietic progenitors, CFUC and CFUE, was studied in vitro. The lipoprotein fractions, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), high density lipoproteins (HDL2 and HDL3), were isolated by sequential ultracentrifugation. The addition of each subfraction to lipoprotein deficient medium allowed us to distinguish two categories of lipoproteins: firstly, those with density d greater than 1.030, LDL, HDL2 and HDL3 which showed a marked inhibitory activity on CFUC and CFUE proliferation and, secondly, those with density d less than 1.030, VLDL and IDL which did not show any inhibitory activity. The regulatory role of lipoproteins on progenitor cell proliferation is discussed.

The role of methylcellulose on colony growth of human myeloid leukemic progenitors (AML-CFU).

The use of a semisolid support like methylcellulose (MC) in a clonogenic assay prevents cell migration and nonspecific aggregation. However, the inhibitory effect of MC on myeloid cell lines has been reported. To assess the effect of MC on human leukemic progenitor cell growth (acute myeloblastic leukemia colony-forming units, AML-CFU), increasing concentrations of MC (0.36%, 0.72%, and 1.44%) were added in a double-feeder culture system. T-lymphocyte-depleted leukemic cells from 12 patients with AML were cultured in the presence of 2.5% phytohemagglutinin (PHA) in a liquid and a semisolid (MC) medium over a leukocyte feeder layer. The leukemic nature of the colonies was confirmed by cytogenetic studies. The median cloning efficiency in the optimal MC assay system was significantly higher (217 leukemic colony-forming units [CFU-L]/5 x 10(4) cells) than the one obtained in the liquid assay system (72.5 CFU-L/5 x 10(4) cells). However, three patterns of growth were observed: 1) colony formation was significantly better in MC than in the liquid assay system (seven of ten cases), 2) there was no difference in growth response (three of ten cases), and 3) colony formation was significantly better in the liquid assay system (one of ten cases). In the semisolid assay system, colony growth was dependent on MC concentration and varied among individual patients. A striking feature was the partial reduction of AML-CFU growth at 1.44% MC, with complete inhibition in 4/11 cases. This phenomenon was not observed for normal progenitors cultured under the same conditions. Cytological evaluation of AML-CFU showed an incomplete maturation to the myelocyte state, accompanied occasionally by macrophagic differentiation. In contrast, maturation of the granulocyte-macrophage colony-forming unit (CFU-GM) clones was harmonious, resulting in greater than 40% polynuclear cells, even from a 7-day culture. Despite a variable clonal response of leukemic progenitors from individual patients, we conclude that 0.72% MC is the optimal concentration of MC in our system, allowing clonal growth of AML-CFU.

Detection of BCR/ABL translocation by polymerase chain reaction in leukemic progenitor cells (ALL-CFU) from patients with acute lymphoblastic leukemia (ALL).

In about 30% of cases, BCR/ABL transcripts are present in freshly isolated mononuclear cells from the bone marrow of patients with acute lymphoblastic leukemia (ALL) using the polymerase chain reaction (PCR) technique. We applied PCR to investigate the leukemic nature of ALL colony-forming unit (ALL-CFU) colonies grown in a clonogenic assay using a double feeder system. The high sensitivity of PCR enables us to detect 1 leukemic cell among 10(5) normal cells. Several controls were taken to assess contamination. In this report we studied three patients with ALL. In all of them, BCR/ABL transcripts were detected at initial diagnosis. We showed that the PCR technique could identify the leukemic nature of ALL-CFU progenitors. In addition, we demonstrated the nonleukemic nature of granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies using the same analysis. We conclude that the PCR technique is of great value in the identification of leukemic clones whenever specific molecular markers are present.

Distinct hematopoietic support by two human stromal cell lines.

OBJECTIVE: The hematopoietic microenvironment is complex, and the role of myofibroblast in its function is crucial. In order to obtain a stable model reflecting this particular cell type, we have previously established human bone marrow cell lines from primary myofibroblastic Stro1(+) population (pStro1(+)). We placed HPV16 E6 and E7 expression under the control of different promoters. Here, we have characterized and studied the hematopoietic support for two cell lines corresponding to the promoters alpha-SM (alphaSM-56 line) and SV40 (SV40-56 line). MATERIALS AND METHODS: The expression profile was analyzed at the RNA level by gene array and at the protein level by Western blot, flow cytometry, and ELISA. Hematopoietic support determined using colony-forming unit (CFU) and stroma-adherent colony-forming cell (SA-CFC) assays. RESULTS: The phenotype of cell lines was not significantly modified compared with primary myofibroblastic cells. They secreted a broad spectrum of hematopoietic cytokines and nonspecific mediators. The two lines allowed the growth of hematopoietic precursors and had different support capabilities. CONCLUSIONS: We have extensively characterized two novel human bone marrow stromal cell lines. They retained a myofibroblastic phenotype and have substantial but different hematopoietic support capabilities. These lines provided a basis for determining stromal factors involved in stem-cell regulation.

Study of granulocyte-macrophage progenitor (CFUc) preservation after slow freezing of bone marrow in the gas phase of liquid nitrogen.

Storage of human hematopoietic stem cells has been made possible through effective preservation of viability by freezing technique. We have studied the quantitative and qualitative aspects of granulocyte-macrophage precursor (CFUc) recovery from 29 bags of frozen bone marrow stored at - 160 degree C in the gas phase of liquid nitrogen for several months or years. it has been shown that with the freezing and preservation technique used, over 75% of the proliferative capacity of the cryopreserved CFUc was recovered. The influence of various factors on the quality of bone marrow preservation was studied and showed that dimethylsulfoxide (DMSO) appeared to cause substantial loss of CFUc at 4 degree C. Evaluation of CFUc in vitro is essential for determining the quality and richness of cryopreserved bone marrow with a view to using such marrow for autologous grafting intensive chemotherapy or total body irradiation in patients with hematological malignancies or solid tumors. No correlation was found between the number of nucleated bone marrow cells and CFUc content.

Recovery of CFU-GM from cryopreserved marrow and in vivo evaluation after autologous bone marrow transplantation are predictive of engraftment.

In 18 patients with non-Hodgkin's lymphomas or solid tumors treated with intensive chemotherapy and/or total-body irradiation followed by autologous bone marrow transplantation (ABMT), we assessed the value of granulocyte-macrophage progenitor cells (CFU-GM) monitoring to predict engraftment. We studied CFU-GM in cryopreserved marrow and attempted to settle whether detection of CFU-GM in vivo after ABMT has a predictive value on engraftment. Our data showed: The absence of linear correlation linking recovery of hematopoiesis to the dose of CFU-GM/kg infused. The existence of a CFU-GM threshold in respect to engraftment. Patients receiving doses of CFU-GM greater than 10(3)/kg had significantly faster recovery kinetics for hematopoiesis than did patients receiving doses below this threshold, with median recoveries to 0.5 and 1.0 X 10(9) neutrophils/liter, respectively, on days 14 and 15 versus days 29 and 31.5 (p less than 0.05 and p less than 0.02) and median recoveries to 1.0 and 2.5 X 10(9) leukocytes/liter respectively, on days 12.5 and 16 versus days 28 and 30.5 (p less than 0.05 and p less than 0.02). Considering the entire course of events during the first four weeks, we were able to show that white blood cell recovery was significantly faster in the group of patients receiving doses of CFU-GM greater than 10(3)/kg (p less than 0.001). Sequential studies of the reappearance of CFU-GM in marrow and peripheral blood indicated that the kinetics of CFU-GM recovery in vivo after ABMT predict engraftment. By day 7 after the graft, CFU-GM were already detectable in the marrow at a level of 10% of the dose infused for patients with optimal engraftment--median time to recovery to 1.0 and 2.5 X 10(9) leukocytes/liter and 1.0 X 10(9) neutrophils/liter on days 11, 15, and 14.5 versus days 18, 23, and 23 (p less than 0.02, less than 0.05, and less than 0.05), respectively after. On day 10 after ABMT, a 15% CFU-GM level in bone marrow confirmed engraftment, with a significant correlation of all parameters studied--1.0 and 2.5 X 10(9) leukocytes/liter (p less than 0.02 and less than 0.01), 0.5 and 1.0 X 10(9) neutrophils/liter (p less than 0.05), 50.0 and 100.0 X 10(9) platelets/liter (p less than 0.05). On day 14, a 50% CFU-GM level was reached in all patients with optimal engraftment; p less than 0.01 on 1.0, and 2.5 X 10(9) leukocytes on 0.5 and 1.0 X 10(9) neutrophils/liter. The detection of circulating CFU-GM in the blood by day 10 or 14 indicated engraftment.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of lymphocyte subsets after autologous bone marrow transplantation with marrow treated by ASTA Z 7557 in acute leukemia: incidence of the in vitro treatment.

The lymphocyte subset reconstitution after high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation (ABMT) has been studied in ten patients with acute leukemia (AL) (6 ALL and 4 ANLL) in complete remission (CR). Bone marrow was treated in vitro with high-dose ASTA Z 7557, individually determined according to CFU-GM sensitivity. The different peripheral blood lymphocyte subsets were characterized by means of monoclonal antibodies (indirect immunofluorescence assay) belonging to the following classes of differentiation: OKT11-T11 (CD2), OKT3-T3 (CD3), OKT4-T4 (CD4), OKT8-T8 (CD8), OKIal-I2 (HLA-DR), Leu7 (natural killer/killer) and by means of polyspecific antiimmunoglobulin sera (direct immunofluorescence assay). Data in these ten patients were compared with those of a control group of 21 normal donors and with a control group of 14 patients in CR without ABMT. Our results showed a marked depression of the T4:T8 ratio in patients with AL before ABMT, compared with normal donors who had respective values of 1.02 and 1.33 (p less than 0.01). This depression was increased and prolonged up to day 515 after ABMT, with a value of 0.32 (p less than 0.01 compared with the pregraft situation; p less than 0.001 compared with normal donors). This T4:T8 ratio imbalance was related to the depletion of the T4+ population and to the increase of the T8+ subset. This imbalance was emphasized after ABMT. The Leu 7+ population was also increased in grafted patients compared with normal donors (p less than 0.01). The B-cell population remained unchanged throughout the study. We conclude that patients autografted with marrow treated in vitro by high-dose ASTA Z 7557 may experience a long-term T-cell subset imbalance.

In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells.

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

Autologous marrow transplantation for patients with chronic myeloid leukemia in accelerated or blastic phase: report of 14 cases.

Between June 1979 and October 1983, 14 autografts were performed in 13 patients with CML (ten blast crisis, four accelerated phase). Results were disappointing: four patients died during aplasia; seven returned to chronic phase, but three died of hemorrhage, four relapsed, and three did not reverse. The main problem was the very low rate of successful engraftment. Both the collection of bone marrow after treatment with busulfan and a particular sensitivity of CFU-GM to cryoinjury were responsible for the infusion of very low doses of CFU-GM. However, we observed some promising results: In one patient in acute blast crisis, the Ph 1 chromosome disappeared, as well as the cytogenetic marker of transformation; in another patient with acute pure cytogenetic acceleration, the abnormal clone disappeared for 27 months; a third patient was maintained in a second chronic phase for 20 months. Thus we suggest that the results of autografting in chronic myeloid leukemia would be improved by infusing the largest possible dose of stem cells collected before or long after treatment by busulfan, and freezing them following a careful program.

Importance of marrow dose on posttransplant outcome in acute leukemia: models derived from patients autografted with mafosfamide-purged marrow at a single institution.

Several prospective randomized trials in acute myelocytic leukemia (AML) documented a lower relapse rate with autologous bone marrow transplantation (ABMT) than with conventional chemotherapy. However, they also identified some transplant difficulties, such as failure to collect sufficient numbers of stem cells, slow kinetics of engraftment, and a high transplant-related mortality that diminished or negated positive impact on overall survival. Data for ABMT are inconclusive in acute lymphocytic leukemia (ALL) in adults. We retrospectively analyzed patients with acute leukemia autografted with marrow purged with mafosfamide after January 1983 in our institution. The population comprised 229 consecutive patients; 165 with AML [123 in first remission (CR1), 32 in second remission (CR2)]; 61 with ALL (46 in CR1, 4 in CR2); and 3 with undifferentiated acute leukemia. All patients were autografted with marrow purged with mafosfamide. Mafosfamide was given at a constant dose of 50 microg/mL in 103 and adjusted individually to produce a CFU-GM LD 95 (5% residual CFU-GM post purging) in 126. The outcome was analyzed for correlation with patient characteristics, the disease including cytogenetics, and the graft itself. Prognostic factors identified by multivariate analysis were used to derive a prognostic classification. Patients receiving higher doses of marrow submitted to purging (>5.46 x 10(4) CFU-GM/kg) experienced a lower treatment-related mortality (RR = 0.11, p = 0.005) and a higher leukemia-free (RR = 0.5, p = 0.005) and overall survival (RR = 0.4, p = 0.001). Patients receiving <0.004% CFU-GM of marrow actually infused post purging had a lower relapse rate (RR = 0.51, p = 0.003). Modeling of prognostic groups identified good-, intermediate-, and poor-risk categories. Patients receiving a stem cell dose evaluated before purging of >5.46 x 10(4) CFU-GM/kg and doses actually infused post purging of < or =0.02 x 10(4)/kg had a treatment-related mortality of only 2+/-2%, a leukemia-free survival of 70%, and an overall survival of 77+/-7% at 10 years. In this study of autotransplantation for acute leukemia using mafosfamide-purged marrow, the stem cell dose used for purging and the intensity of purging were the most important factors predicting outcome.