We combined two experimental approaches — a transplantation model of human fetal pancreas that recapitulates development of human pancreatic islets and lentivirus-mediated gene transfer — to demonstrate that beta cells from a single human islet are derived from multiple progenitors.
In mice, one previous study has used chimeras to demonstrate that each islet consists of a mixture of beta cells originating from a pool of progenitor cells 
, but this has not been determined for human islets. Overall, information on pancreas development, beta-cell differentiation and islet morphogenesis in human pancreas is rare compared to rodent 
. This is due to limited access to human tissues, as well as to lack of experimental systems to study human pancreatic development. It is currently believed that human and rodent islets develop the same way. Thus, it has been suggested that beta cells may be generated from human embryonic stem cells, based on our knowledge of rodent pancreatic development 
. However, rodent and human islets share many similarities, but also differ substantially. For example, cell proliferation, susceptibility to destruction and beta-cell function differ between rodents and humans 
. Moreover, islet cytoarchitecture, possibly with functional implications, also differs 
Our knowledge of human pancreatic development is mainly based on previous studies comparing human pancreatic sections taken at various stages of development 
. However, tissue sections are not informative in determining whether beta cells from a single islet develop from one or several progenitors. Nor is the aggregation chimera system used for rodents 
applicable to address this issue in humans. In fact, few dynamic models mimicking islet cell development are available 
. Here, we developed a novel experimental system based on lentivirus-mediated gene transfer in a reconstituted model of human pancreatic development. We previously showed that development of human fetal pancreas transplanted under the kidney capsule of immuno-incompetent scid
mice reflects that of normal human pancreas development 
. Within the graft endocrine cells differentiate and functional human islets of Langerhans containing all endocrine cell types are formed after a few months 
. In this study, we showed that the cytoarchitecture of human islets that developed ex vivo
in the scid
mice is similar to that of normal adult human islets.
During pancreatic development in rodents, beta cells are derived from Pdx1-positive progenitors with a high proliferative potential 
. When the cells enter the endocrine pathway and start to express the transcription factor Neurogenin3, their proliferation rate decreases sharply 
. During the later perinatal stage, beta cells derived from Neurogenin3 progenitors and located within the islets reenter the cell cycle 
. Thus, the number of insulin-positive cells present in a rodent islet is probably dependent on the proliferation rates of both progenitor and mature beta cells. We reasoned that if the beta cells present in a single human islet originate from a unique progenitor, Pdx1-positive progenitor cells and/or mature beta cells should proliferate. We found that a large number of Pdx1-positive progenitor cells stained positive for Ki67 10 days after transplantation, demonstrating the proliferative potential of such cells. We also found that some of the beta cells present in human islets that developed in scid
mice were proliferative. The low rate of beta-cell proliferation is consistent with beta-cell proliferation rates found in prenatal human pancreas development 
. Thus, with such data, we could not exclude the possibility that beta cells present in a single islet may originate from a unique progenitor that proliferates at the progenitor stage and/or at the beta-cell stage.
Here, we used low multiplicity of infection to transduce human fetal pancreases with lentiviruses expressing GFP under the control of the rat insulin II promoter. This promoter has previously been used for lentivirus-mediated transgene expression restricted to rodent beta cells 
. Our findings described here demonstrate that restricted expression is also obtained in mature human beta cells. We generated heterogeneous islets composed of some beta cells positive and others negative for the transgene product by transducing a limited number of human fetal pancreatic progenitors and consequently conclude that human islets derive from more than one progenitor.
Little is known about tissue morphogenesis in humans mainly due to the absence of informative experimental models. In this study, we developed a unique model combining ex vivo organogenesis from human fetal tissue and cell type-specific lentivirus-mediated gene transfer. This approach resulted in the generation of human transgenic-like developing tissue. This original model allowed a dynamic analysis of human islet formation during development. It can now be generalized as a powerful transgenic-like tool to examine in a dynamic fashion the genetic processes involved in tissue formation in humans.