Células mesenquimales de médula osea: diferenciación y potencial reemplazo neuronal / Mesenchymal stem cells: differentiation and alternative source of neural tissue

Embryonic stem cells are a population of cells located in the blastocyst, committed to specific differentiation according to spatial and temporal factors such as age and place of final location. Despite the final fate of hematic cells, hemopoietic cells retain a relative degree of plasticity dependent on environmental factors. Mesenchymal cells are a well differentiated population of bone marrow derived non hemopoietic cells with totipotential properties. The medical interest of such totipotentiality rests in the potential of such cells to repair damaged tissues. Particularly neuronal differentiation from progenitors obtained from mesenchymae non hemopoietic cells offers a new possibility in the field of neural transplantation and tissue engineering to repair functional entities in the nervous system.

Las células troncales embrionarias son totipotentes y se encuentran en pequeño número en el blastocisto donde pueden expandirse en forma indiferenciada durante un corto tiempo y de acuerdoal sitio donde se alojan, ellas adquirirán determinados fenotipos de diferenciación. Las células hemopoyéticas troncales se caracterizan por poseer un gran potencial proliferativo, cuyo modelo de regulación es jerárquico. Ellas retienen, a lo largo de su existencia, un cierto grado de plasticidad, lo cual hace que se puedan diferenciar en distintos tipos de células o de tejidos no hemopoyéticos, de acuerdo al microambiente donde se encuentran o bien a la presencia de ciertos factores estimulantes. En trabajos recientes se han podido aislar, por adherencia al plástico, en cultivo in vitro de médula ósea, células no hemopoyéticas que fueron llamadas mesenquimales por su semejanza con el tejido mesenquimal del embrión. Estas células, in vitro, pueden ser inducidas hacia nuevas líneas celulares, que se diferenciarán en nuevos tejidos. Las expectativas del beneficio terapéutico del transplante de médula ósea como el de células mesenquimales en enfermedades no hemopoyéticas son grandes, porque al utilizar tejidos o células autólogas, se evitan los graves problemas del rechazo inmunológico. En el caso del tejido nervioso la problemática de una fuente adecuada de donantes hace el tema especialmente interesante.

[Characteristics of murine primordial germ cells and their relation with hemopoiesis]. / Características de las células primordiales germinales murinas y su relación con la hemopoyesis.

Primordial germ cells (PGC) are a population of cells characterised by a positive reaction to alkaline phosphatase, usually present in the mouse embryo at 7.5 days post coitus (dpc). These cells migrate through various tissues before they become incorporated into the gonadal ridges. Hematopoiesis is a complex developmental system in which the hemopoietic stem cells (HSC) were experimentally shown to have been derived from a single multipotent stem cell. PGC, as well as HSC are regulated by a range of growth factors that control both proliferative and differentiative processes. Leukemia inhibitory factor (LIF) is a cytokine that regulates the differentiation and the totipotentional phenotype of PGC. Recently, other growth factors, such as stem cell factor (SCF), macrophage growth factor (MGF), and forskolin (FRKL) have been proposed as the possible in vivo and in vitro regulators for PGCs and HSCs. Induction of hematopoiesis in an embryonic germ cell derived from PGCs indicates that germ cells acquire the potentiality to differentiate toward hematopoietic cells. The coincidental presence of both PGCs and HSCs at the sites where early hemopoiesis is established, together with similar growth factor requirements support the hypothesis that PGCs may also be considered hemopoiesis initiating cells.

[Analysis of hemopoietic progenitor cells in bone marrow and peripheral blood samples which have undergone different periods of cryopreservation]. / Analisis de progenitores hemopoyéticos en muestras de medula osea y de sangre periférica en periodos variables de criopreservación.

Bone marrow (BM) and peripheral blood stem cell (PBSC) samples from patients undergoing autologous transplant were tested to evaluate the effects of cryopreservation. Cell viability was assessed as well as the proliferative capability of CFU-GM and BFU-E (myeloid and erythroid progenitors respectively). Moreover, long term culture (LTC) of stromal cells was used to test their functionality. A total of 23 samples were studied: 5 from AML patients, 7 MM, 6 NHL, 3 ALL and 2 HL. Nine patients received autologous bone marrow transplant (ABMT) and the remaining 14 PBSC. The cells were frozen during 24 to 33 days before infusion and 16 to 40 months before culture. Forty percent of AML and MM samples gave rise to colonies in vitro while the other hematology diseases tested showed colony growth in almost 100% of the cases. Samples from patients with lymphoid diseases exhibited a good correlation between the percentage of CD34+ cells and the number of colonies developed in culture. Nevertheless, there was no correlation when ALL and MM were tested suggesting that the underlying disease may have affected the growth in culture. The stromal layer was fully developed on BM samples, but on PBSC samples it only generated macrophages and fibroblasts. Our results suggest that the efficacy of cryopreservation of hematopoietic cells can be measured by means of CFU-GM and BFU-E culture and that the period the samples remained frozen did not affect the growth capability of the cells.

[In vitro study of hemopoietic progenitors in human umbilical cord blood treated with different cytokines and autologous serum]. / Estudio in vitro de progenitores hemopoyéticos de sangre de cordón umbilical humano frente a diferentes citoquinas y suero autólogo.

UNLABELLED: The aim was to obtain the ex vivo expansion of human umbilical cord blood (HUCB) cells. A total of 19 samples were assayed to evaluate the number and type of hemopoietic progenitor cells, their proliferating capacity and the stimulating potency of cord blood serum. METHODS: a) CFU-GM, CFU-GEMM and BFU-E cultures in the presence of CSF, BM serum or HUCB serum; b) 35 day LTC in liquid media whether in presence of IL-3 + GM-CSF + SCF or autologous serum (AS). Cells were demidepopulated at 7-day intervals and fresh medium and cytokines were added. Harvested cells were cultured in bone marrow (BM)/ SM and colonies were evaluated after 10 days. RESULTS: The mean number of CFU-GM was similar to BM values; the maximum number of colonies was observed at day 7 and remained high until day 21 whether in addition of cytokines or AS. A total of 8 samples gave rise to colonies up to day 35; these samples showed higher values than BM in SM; HUCB serum has a great stimulating effect on BM cells and HUCB cells compared with nonspecific stimulating factors; moreover, HUCB showed a large dispersion. CONCLUSION: 1) HUCB contains a high number of hemopoietic progenitor cells with a large dispersion coefficient, 2) HUCB plasma has a great stimulatory capacity, 3) it is possible to induce the expansion of HUCB progenitors in LTC either in the presence of cytokines or of AS without loss of potency.

[Hematopoiesis regulation]. / Regulación de la hematopoyesis.

Positive and negative signals are crucial in the regulation of the hematopoietic system. In the last 30 years, more than 20 molecules (glycoproteins) with biological activity upon the hematopoietic progenitor cells and even on the mature blood cells have been purified. The best known of these biomolecules are the hematopoietic growth factors (colony stimulating factors and interleukins), which are able to stimulate bone marrow cells to give mature progeny. At present, not only the sequence of the majority of these glycoproteins and their codifying genes has been determined, but also their target cells and cellular receptors. Research studies of the interaction between the hematopoietic progenitor cells and their stimulating and inhibiting factors are very helpful in the development of clinical trials and have become important tools to explore the origin of a great number of hematological diseases. However, the mechanisms underlying growth/inhibitory factor production and progenitor cell proliferation remain poorly understood.

The myelodysplastic syndrome: prognostic factors in relation to the "in vitro" clonogenic assay.

The object of this study was to determine whether the "in vitro" parameters of medullary and blood granulopoiesis in patients with MDS, furnish information of either prognostic or diagnostic value. This study covered 94 patients with MDS. All patients were studied at the onset of disease. In order to identify the factors related to patients' survival, Cox Multiple Regression analysis was performed by the BMD P2L program. When analyzing by means of actuarial curves the survival probability of patients with benign development versus those of malignant development (those who developed ANLL), the significance between both groups was p = 0.0001. Different variables of patients included in this study were analyzed and all showed great significances. Fab: p = 0.0022, disease evolution: p = 0.0001 and presence of blastic aggregates: p = 0.0011. Cox's regression analysis revealed that the only predictable survival variable is the presence of blastic colonies and/or clusters. Accordingly, two groups were constructed: favourable and unfavourable. In the favourable group, 40% of the patients belonged to the RA group, while in the unfavourable group, 55% belonged to the RAEB group. This study shows the validity of the elaboration of prognostic groups in MDS according to the presence of blastic colonies and/or clusters in CFUGM medullary and/or peripheral cultures. The "in vitro" myeloid progenitors culture techniques may therefore be advantageously applied in these disorders for formulating a diagnosis and predicting the patient's short term evolution.