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Objective: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. Methods: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients, respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. Results: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (≥98%) for CD73, CD105, CD106, CD29, CD13, CD44 and negative (≤1%) for CD34, CD45, CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients, respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. Conclusions: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. © 2006 International Society for Experimental Hematology.

Objective Intravenous (IV) injection is currently the normal method for transplanting hematopoietic cells. However, the problem of seeding efficiency and homing is relevant especially when a limited number of stem cells is available. Intra–bone marrow (IBM) injection of bone marrow cells (BMCs) may overcome this problem. Materials and methods Irradiated (750 cGy) C57BL/6J mice were transplanted with 1 × 10 5 BMCs harvested from transgenic mice expressing an enhanced version of the green fluorescent protein (EGFP + ) via IBM or with 1 × 10 6 EGFP + BMCs via IV. Irradiated (320 cGy) NOD/SCID mice were transplanted with 1 × 10 6 human cord blood (CB) cells via IBM or with 1 × 10 7 human CB cells via IV. Results In C57BL/6J mice after 90 days, the fraction of EGFP + cells harvested was 37% and 53% in IV-treated and IBM-treated (contralateral tibia and femur in the IBM) mice, respectively: the expansion folds were 114 and 1760, respectively. In NOD/SCID mice, the percentages of CD45 + cells and CD45 + /CD34 + cells were, at 30 days, 3.3% and 0.3% in IV-treated mice, and 4.4% and 1.1% in IBM-treated mice. At 60 days, the percentages of CD45 + cells and CD45 + /CD34 + cells were 2.1% and 0.3% in IV-treated mice and 1.4% and 0.4% in IBM-treated mice. At day 90 the percentages of CD45 + cells and CD45 + /CD34 + cells were 3% and 0.3% in IV-treated mice and 2.3% and 0.4% in IBM-treated mice. Conclusion Our data demonstrate that IBM transplantation is associated with a seeding efficiency 15 times greater than IV transplantation. IBM transplantation may improve the results of transplant and may be useful in several settings: 1) when a limited number of hematopoietic progenitors are available; and 2) in experiments aiming to place in the bone marrow stem cells of other lineages (CNS, muscle, etc.).

+HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However, recent studies demonstrated, by RT-PCR, that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation.We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM, BFU-E, and CFU-GEMM) and early progenitors (LTC-IC) was evaluated.The HOXC4 protein is indeed expressed in human CD34(+) cells, and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold, range 2.5-35; p0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13, onefold; range 4.1-102.1, p0.0001).We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p0.009) in the number of erythroid colonies compared with CFU-GM, although without perturbing, at least in vitro, the maturation program of the cells. On the other hand, the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage.

Objective In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myelo-reductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. Materials and Methods We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%–100% Ph-negative PBPC for a median time of 28 months (range 18–50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). Results Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%–100% to 0%–25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21–43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term- culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p 0.03). Conclusion We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.

Objective To compare the suppressive effect of mesenchymal stem cells (MSC), derived from normal individuals or severe aplastic anemia patients (SAA), on T-cell activation. Patients and Methods We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n=3), following immunosuppressive therapy (IS) (n=16), or after a bone marrow transplant (BMT) (n=4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR), phytohemaglutinin (PHA)-primed cultures, activation surface markers, γ-IFN production, hematopoietic colony formation (CFC), production of cyclic ADP–ribose (cADPR). Results The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens ( p =0.009) or PHA ( p =0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells ( p =0.001); 3) impaired ability to suppress γ-IFN production in PHA cultures, resulting in an 11-fold higher γ-IFN concentration; 4) no preventive effect on T cell–mediated inhibition of CFC; and 5) significantly reduced ( p =0.009) production of cADPR, a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. Conclusion The ability of MSC to downregulate T-cell priming, proliferation, and cytokine release is deficient in patients with SAA, persists indefinitely after immunosuppressive therapy, but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined.

The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood.We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4).The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p10(-4) and p0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p0.02 and p0.001 respectively).Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

Objective. An objective of autologous transplant in chronic myelogenous leukemia is to reinfuse the patient stem cells with the lowest amount of leukemic progenitors. We analyzed if the cytogenetic response after autografting was correlated with the amount of BCR ABL–positive cells present in the graft. Materials and Methods. By BCR-ABL mRNA quantification, we studied the serial pheresis product from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplant in our unit. Therefore, we correlated the residual disease in the graft reinfused to the patients with the cytogenetic response after autografting, taking into consideration only those responses that lasted 12 months or more. Results. Thirty-two patients had a graft with 0–35% Ph-metaphases and 19 had a graft with BCR-ABL/ABL ratio ≤0.01. After a median of 27 months (range, 12–50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months and 14 of them (78%) had a graft with BCR-ABL/ABL ratio ≤0.01 (range, 0.0003–0.01). Twenty-two patients had short-lived response or showed to be >35% Ph-positive in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio ≤0.01 (range, 0.001–0.01). Therefore, we found a strong association between BCR-ABL/ABL ratio ≤0.01 in the reinfused progenitor collections and the achievement of complete or major cytogenetic remission after autografting ( χ 2 test, p = 0.0001). Patients reinfused with low contaminated grafts also showed a longer duration of the response (logrank test, P = 0.0009). Eleven patients were reinfused with the lowest-contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients had prolonged neutropenia or thrombocytopenia following stem cell reinfusion and 9 of them had long-lasting complete or major cytogenetic response after transplant. Conclusion. This study demonstrates that the amount of BCR-ABL positive cells present in the graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting.