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Hematopoiesis undergoes several migrations from yolk sac to liver and spleen, and finally bone marrow until the end of life. A number of investigations have demonstrated that the hematopoietic microenvironment plays very important role in this process. However, the exact mechanisms remain unknown. In order to systematically analyze and understand the role of hematopoietic microenvironment in the regulation and control of hematopoiesis, a microarray containing 588 complementary DNAs was used to compare the gene expressions between those in murine fetal liver and bone marrow cells. The results obtained from array hybridization were analyzed and reconfirmed by using bioinformatics and RT-PCR as well as Northern blot. The results showed that 65 and 131 genes were relatively high expressed in bone marrow and fetal liver cells respectively among 588 known genes in the array-membrane. According to the survey in the PubMed, 39 out of bone-marrow-expressed genes and 71 in fetal-liver-expressed genes were closely related to the hematopoiesis. Further reconfirmation by RT-PCR or Northern blot has demonstrated that CD18, CD44 an d PSGL-1 genes chosen for analysis were highly expressed in adult bone marrow, but unexpressed or lower expressed in fetal liver cells, resulting in high similarity to the array results. Moreover, the expressions of CD18 and CD44 in fetal liver were down-regulated with the increment of gestational age. In conclusion, the gene expressions in bone marrow and fetal liver cells are obviously different, some of the genes are down-regulated at the different stages of ontogeny. The different gene expression levels between bone marrow and fetal liver, especially those genes closely related to the hematopoiesis, may be the molecular basis for the explanation of why hematopoietic stem cells derived from different tissues have different characterizations as well as the differences from the beginning and terminating of fetal liver hematopoiesis, and why hematopoietic stem cells derived from fetal liver is tremendously difficult to be grafted in bone marrow.

Hematopoietic stromal cells, being the essential ingredient of the hematopoietic microenvironment, play very important roles in the control and regulation of self-renewal, proliferation and differentiation of hematopoietic stem cells (HSC) via complex interactions of cell-cell, cell-humoral and cell-extracellular matrix. Evidence from in vivo experiment has proved that HSC derived from normal mice could reconstitute hematopoiesis of mice with HSC defects but failed to reconstitute hematopoiesis of those mice with microenvironment defects, showing the importance of hematopoietic microenvironment in the maintenance of hematopoiesis in vivo. A well-known long-term culture (LTC) system established by Dexter demonstrated in another way that stromal cell layer in the system could support ex vivo hematopoiesis for several months, even more than one year under the optimal conditions. It, however, has not been demonstrated that what is the key elements and in which way the ex vivo hematopoiesis could be maintained for so long time. As the inventions for the large-scale screening methodologies the suppression subtractive hybridization (SSH) was chosen for the screening differentially expressed genes expressed by LTC cultured stromal cells but not by the uncultured bone marrow cells (BMC). mRNA extracted from both cultured adherent cells (tester) and BMC (driver) were hybridized according to the protocol provided by CLONTECH. Total of 130 clones differentially expressed by cultured cells were randomly picked up and 106 ESTs were obtained after sequencing. They represent 26 identical or similar genes and 7 novel genes after the bioinformatics analysis. 5 of the novel genes with the entire open reading frame, without functional clues, have been cloned into the mammalian expression vectors and the functions of them in the control of proliferation and differentiation of HSC will be further exploring. The most interesting discovery is that 3 novel genes have signal peptides, implying the potential discovery of novel growth factors as 80% known growth factors have signal peptides. Our experimental results suggest that: (a) based on the results of subtractive efficiency, the SSH could be a reliable method to screen differentially expressed genes; (b) gene expression may be regulated by multiple factors, even conditioning-dependent, in this experiment the genes expressed by bone marrow stromal cells are LTC-cultivation inducible; (c) it is possible to find interesting genes or special gene after relatively large-scale screen.